Chapter 13Gene Regulation

in Eukaryotes

Chapter 13Gene Regulation

in Eukaryotes

Eukaryotic gene regulation occurs at several levels

Small percentages of newly synthesized DNAs (~3 in mammals) are chemically modified by methylation

Methylation occurs most often in symmetrical CG sequences

Transcriptionally active genes possess significantly lower levels of methylated DNA than inactive genes

Methylation results in a human disease called fragile X syndrome FMR-1 gene is silenced by methylation

1- Control at DNA level by DNA methylation

bull Acetylation ( 乙酰化 )by histone acetyl transferases (HATs) and coactivators leads to euchromatin formation p53 acetylation

bull Methylation by HDACs （去乙酰化酶） and corepressors leads to heterochromatin formation Rb-E2F

2- Control at DNA level by Histone 2- Control at DNA level by Histone modifications(Chromatin modifications(Chromatin Remodeling)Remodeling)

2- Control at DNA level by Histone 2- Control at DNA level by Histone modifications(Chromatin modifications(Chromatin Remodeling)Remodeling)

3-Control at DNA level by gene 3-Control at DNA level by gene amplificationamplification

Repeated rounds of DNA replication yield multiple copies of a particular chromosomal region

4- Control at transcription 4- Control at transcription initiationinitiation

gene control region for gene X

By using different sequences (promoter enhancer or silencer sequences) and factors the rate of transcription of a gene is controlled

By using different sequences (promoter enhancer or silencer sequences) and factors the rate of transcription of a gene is controlled

Calcitonin gene-related peptide

61

5- Control at mRNA splicing 5- Control at mRNA splicing (alternate splicing)(alternate splicing)

(four exons)

32 amino acidsReduces bone resorption

37 amino acidsVasodilator

bull1048766Messenger RNA longevity can be influenced by several factorsbull1048766Poly(A) tails seem to stabilize mRNAsbull1048766The sequence of the 3rsquountranslated region (3rsquoUTR) preceding a poly(A) tail also seems to affect mRNA stabilitybull1048766Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3rsquountranslated regions

bull1048766Messenger RNA longevity can be influenced by several factorsbull1048766Poly(A) tails seem to stabilize mRNAsbull1048766The sequence of the 3rsquountranslated region (3rsquoUTR) preceding a poly(A) tail also seems to affect mRNA stabilitybull1048766Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3rsquountranslated regions

6- Control at mRNA stability6- Control at mRNA stability

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

6- Control at mRNA stability6- Control at mRNA stability

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

6- Control at mRNA stability6- Control at mRNA stability

7- Control at initiation of translation7- Control at initiation of translation

5rsquo UTR

3rsquo UTRAUG UAA

Specific sequences make specific secondary structures

Specific protein factors bind to these secondary structures

COOH+NH2

NH2

ATP

CO NH

CO NH

ubiquitin protein ligase

Doomed protein

molecule

26S proteaso

me

bullUbiquitin-dependent proteolysis bullProtein molecule is tagged for degradation by attachment of a 20 kDa protein ubiquitin

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

8-Regulation by protein stability8-Regulation by protein stability

Similarity of regulation between eukaryotes and prokaryote

1Principles are the same signals (信号 ) activators and repressors (激活蛋白和阻遏蛋白 )recruitment and allostery cooperative binding (招募异构和协同结合 )

2 The gene expression steps subjected to regulation are similar and the initiation of transcription is the most pervasively regulated step

Difference in regulation between eukaryotes and prokaryote

1 Pre-mRNA splicing adds an important step for regulation (mRNA前体的剪接 )

2 The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart (真核转录机器更复杂 )

3 Nucleosomes and their modifiers influence access to genes (核小体及其修饰体 )

4 Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes (真核基因有更多结合位点 )

15

A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration

Bacteria

Yeast

Human

16

Promoter

Core promoter in eukaryote TATA-box Initiator (Inr) in prokaryote -10 region Inr

Proximal elements of promoter in prokaryote -35 region in eukaryote CAAT-box GC-box UPE upstream promoter element UAS upstream activating sequence Terminator ( 终止子 ) A DNA sequence just

downstream of the coding segment of a gene which is recognized by RNA polymerase as a signal to stop transcription

Cis-acting elementCis-acting element

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Eukaryotic gene regulation occurs at several levels

Small percentages of newly synthesized DNAs (~3 in mammals) are chemically modified by methylation

Methylation occurs most often in symmetrical CG sequences

Transcriptionally active genes possess significantly lower levels of methylated DNA than inactive genes

Methylation results in a human disease called fragile X syndrome FMR-1 gene is silenced by methylation

1- Control at DNA level by DNA methylation

bull Acetylation ( 乙酰化 )by histone acetyl transferases (HATs) and coactivators leads to euchromatin formation p53 acetylation

bull Methylation by HDACs （去乙酰化酶） and corepressors leads to heterochromatin formation Rb-E2F

2- Control at DNA level by Histone 2- Control at DNA level by Histone modifications(Chromatin modifications(Chromatin Remodeling)Remodeling)

2- Control at DNA level by Histone 2- Control at DNA level by Histone modifications(Chromatin modifications(Chromatin Remodeling)Remodeling)

3-Control at DNA level by gene 3-Control at DNA level by gene amplificationamplification

Repeated rounds of DNA replication yield multiple copies of a particular chromosomal region

4- Control at transcription 4- Control at transcription initiationinitiation

gene control region for gene X

By using different sequences (promoter enhancer or silencer sequences) and factors the rate of transcription of a gene is controlled

By using different sequences (promoter enhancer or silencer sequences) and factors the rate of transcription of a gene is controlled

Calcitonin gene-related peptide

61

5- Control at mRNA splicing 5- Control at mRNA splicing (alternate splicing)(alternate splicing)

(four exons)

32 amino acidsReduces bone resorption

37 amino acidsVasodilator

bull1048766Messenger RNA longevity can be influenced by several factorsbull1048766Poly(A) tails seem to stabilize mRNAsbull1048766The sequence of the 3rsquountranslated region (3rsquoUTR) preceding a poly(A) tail also seems to affect mRNA stabilitybull1048766Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3rsquountranslated regions

bull1048766Messenger RNA longevity can be influenced by several factorsbull1048766Poly(A) tails seem to stabilize mRNAsbull1048766The sequence of the 3rsquountranslated region (3rsquoUTR) preceding a poly(A) tail also seems to affect mRNA stabilitybull1048766Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3rsquountranslated regions

6- Control at mRNA stability6- Control at mRNA stability

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

6- Control at mRNA stability6- Control at mRNA stability

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

6- Control at mRNA stability6- Control at mRNA stability

7- Control at initiation of translation7- Control at initiation of translation

5rsquo UTR

3rsquo UTRAUG UAA

Specific sequences make specific secondary structures

Specific protein factors bind to these secondary structures

COOH+NH2

NH2

ATP

CO NH

CO NH

ubiquitin protein ligase

Doomed protein

molecule

26S proteaso

me

bullUbiquitin-dependent proteolysis bullProtein molecule is tagged for degradation by attachment of a 20 kDa protein ubiquitin

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

8-Regulation by protein stability8-Regulation by protein stability

Similarity of regulation between eukaryotes and prokaryote

1Principles are the same signals (信号 ) activators and repressors (激活蛋白和阻遏蛋白 )recruitment and allostery cooperative binding (招募异构和协同结合 )

2 The gene expression steps subjected to regulation are similar and the initiation of transcription is the most pervasively regulated step

Difference in regulation between eukaryotes and prokaryote

1 Pre-mRNA splicing adds an important step for regulation (mRNA前体的剪接 )

2 The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart (真核转录机器更复杂 )

3 Nucleosomes and their modifiers influence access to genes (核小体及其修饰体 )

4 Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes (真核基因有更多结合位点 )

15

A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration

Bacteria

Yeast

Human

16

Promoter

Core promoter in eukaryote TATA-box Initiator (Inr) in prokaryote -10 region Inr

Proximal elements of promoter in prokaryote -35 region in eukaryote CAAT-box GC-box UPE upstream promoter element UAS upstream activating sequence Terminator ( 终止子 ) A DNA sequence just

downstream of the coding segment of a gene which is recognized by RNA polymerase as a signal to stop transcription

Cis-acting elementCis-acting element

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Small percentages of newly synthesized DNAs (~3 in mammals) are chemically modified by methylation

Methylation occurs most often in symmetrical CG sequences

Transcriptionally active genes possess significantly lower levels of methylated DNA than inactive genes

Methylation results in a human disease called fragile X syndrome FMR-1 gene is silenced by methylation

1- Control at DNA level by DNA methylation

bull Acetylation ( 乙酰化 )by histone acetyl transferases (HATs) and coactivators leads to euchromatin formation p53 acetylation

bull Methylation by HDACs （去乙酰化酶） and corepressors leads to heterochromatin formation Rb-E2F

2- Control at DNA level by Histone 2- Control at DNA level by Histone modifications(Chromatin modifications(Chromatin Remodeling)Remodeling)

2- Control at DNA level by Histone 2- Control at DNA level by Histone modifications(Chromatin modifications(Chromatin Remodeling)Remodeling)

3-Control at DNA level by gene 3-Control at DNA level by gene amplificationamplification

Repeated rounds of DNA replication yield multiple copies of a particular chromosomal region

4- Control at transcription 4- Control at transcription initiationinitiation

gene control region for gene X

By using different sequences (promoter enhancer or silencer sequences) and factors the rate of transcription of a gene is controlled

By using different sequences (promoter enhancer or silencer sequences) and factors the rate of transcription of a gene is controlled

Calcitonin gene-related peptide

61

5- Control at mRNA splicing 5- Control at mRNA splicing (alternate splicing)(alternate splicing)

(four exons)

32 amino acidsReduces bone resorption

37 amino acidsVasodilator

bull1048766Messenger RNA longevity can be influenced by several factorsbull1048766Poly(A) tails seem to stabilize mRNAsbull1048766The sequence of the 3rsquountranslated region (3rsquoUTR) preceding a poly(A) tail also seems to affect mRNA stabilitybull1048766Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3rsquountranslated regions

bull1048766Messenger RNA longevity can be influenced by several factorsbull1048766Poly(A) tails seem to stabilize mRNAsbull1048766The sequence of the 3rsquountranslated region (3rsquoUTR) preceding a poly(A) tail also seems to affect mRNA stabilitybull1048766Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3rsquountranslated regions

6- Control at mRNA stability6- Control at mRNA stability

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

6- Control at mRNA stability6- Control at mRNA stability

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

6- Control at mRNA stability6- Control at mRNA stability

7- Control at initiation of translation7- Control at initiation of translation

5rsquo UTR

3rsquo UTRAUG UAA

Specific sequences make specific secondary structures

Specific protein factors bind to these secondary structures

COOH+NH2

NH2

ATP

CO NH

CO NH

ubiquitin protein ligase

Doomed protein

molecule

26S proteaso

me

bullUbiquitin-dependent proteolysis bullProtein molecule is tagged for degradation by attachment of a 20 kDa protein ubiquitin

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

8-Regulation by protein stability8-Regulation by protein stability

Similarity of regulation between eukaryotes and prokaryote

1Principles are the same signals (信号 ) activators and repressors (激活蛋白和阻遏蛋白 )recruitment and allostery cooperative binding (招募异构和协同结合 )

2 The gene expression steps subjected to regulation are similar and the initiation of transcription is the most pervasively regulated step

Difference in regulation between eukaryotes and prokaryote

1 Pre-mRNA splicing adds an important step for regulation (mRNA前体的剪接 )

2 The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart (真核转录机器更复杂 )

3 Nucleosomes and their modifiers influence access to genes (核小体及其修饰体 )

4 Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes (真核基因有更多结合位点 )

15

A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration

Bacteria

Yeast

Human

16

Promoter

Core promoter in eukaryote TATA-box Initiator (Inr) in prokaryote -10 region Inr

Proximal elements of promoter in prokaryote -35 region in eukaryote CAAT-box GC-box UPE upstream promoter element UAS upstream activating sequence Terminator ( 终止子 ) A DNA sequence just

downstream of the coding segment of a gene which is recognized by RNA polymerase as a signal to stop transcription

Cis-acting elementCis-acting element

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

bull Acetylation ( 乙酰化 )by histone acetyl transferases (HATs) and coactivators leads to euchromatin formation p53 acetylation

bull Methylation by HDACs （去乙酰化酶） and corepressors leads to heterochromatin formation Rb-E2F

2- Control at DNA level by Histone 2- Control at DNA level by Histone modifications(Chromatin modifications(Chromatin Remodeling)Remodeling)

2- Control at DNA level by Histone 2- Control at DNA level by Histone modifications(Chromatin modifications(Chromatin Remodeling)Remodeling)

3-Control at DNA level by gene 3-Control at DNA level by gene amplificationamplification

Repeated rounds of DNA replication yield multiple copies of a particular chromosomal region

4- Control at transcription 4- Control at transcription initiationinitiation

gene control region for gene X

By using different sequences (promoter enhancer or silencer sequences) and factors the rate of transcription of a gene is controlled

By using different sequences (promoter enhancer or silencer sequences) and factors the rate of transcription of a gene is controlled

Calcitonin gene-related peptide

61

5- Control at mRNA splicing 5- Control at mRNA splicing (alternate splicing)(alternate splicing)

(four exons)

32 amino acidsReduces bone resorption

37 amino acidsVasodilator

bull1048766Messenger RNA longevity can be influenced by several factorsbull1048766Poly(A) tails seem to stabilize mRNAsbull1048766The sequence of the 3rsquountranslated region (3rsquoUTR) preceding a poly(A) tail also seems to affect mRNA stabilitybull1048766Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3rsquountranslated regions

bull1048766Messenger RNA longevity can be influenced by several factorsbull1048766Poly(A) tails seem to stabilize mRNAsbull1048766The sequence of the 3rsquountranslated region (3rsquoUTR) preceding a poly(A) tail also seems to affect mRNA stabilitybull1048766Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3rsquountranslated regions

6- Control at mRNA stability6- Control at mRNA stability

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

6- Control at mRNA stability6- Control at mRNA stability

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

6- Control at mRNA stability6- Control at mRNA stability

7- Control at initiation of translation7- Control at initiation of translation

5rsquo UTR

3rsquo UTRAUG UAA

Specific sequences make specific secondary structures

Specific protein factors bind to these secondary structures

COOH+NH2

NH2

ATP

CO NH

CO NH

ubiquitin protein ligase

Doomed protein

molecule

26S proteaso

me

bullUbiquitin-dependent proteolysis bullProtein molecule is tagged for degradation by attachment of a 20 kDa protein ubiquitin

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

8-Regulation by protein stability8-Regulation by protein stability

Similarity of regulation between eukaryotes and prokaryote

1Principles are the same signals (信号 ) activators and repressors (激活蛋白和阻遏蛋白 )recruitment and allostery cooperative binding (招募异构和协同结合 )

2 The gene expression steps subjected to regulation are similar and the initiation of transcription is the most pervasively regulated step

Difference in regulation between eukaryotes and prokaryote

1 Pre-mRNA splicing adds an important step for regulation (mRNA前体的剪接 )

2 The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart (真核转录机器更复杂 )

3 Nucleosomes and their modifiers influence access to genes (核小体及其修饰体 )

4 Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes (真核基因有更多结合位点 )

15

A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration

Bacteria

Yeast

Human

16

Promoter

Core promoter in eukaryote TATA-box Initiator (Inr) in prokaryote -10 region Inr

Proximal elements of promoter in prokaryote -35 region in eukaryote CAAT-box GC-box UPE upstream promoter element UAS upstream activating sequence Terminator ( 终止子 ) A DNA sequence just

downstream of the coding segment of a gene which is recognized by RNA polymerase as a signal to stop transcription

Cis-acting elementCis-acting element

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

3-Control at DNA level by gene 3-Control at DNA level by gene amplificationamplification

Repeated rounds of DNA replication yield multiple copies of a particular chromosomal region

4- Control at transcription 4- Control at transcription initiationinitiation

gene control region for gene X

By using different sequences (promoter enhancer or silencer sequences) and factors the rate of transcription of a gene is controlled

By using different sequences (promoter enhancer or silencer sequences) and factors the rate of transcription of a gene is controlled

Calcitonin gene-related peptide

61

5- Control at mRNA splicing 5- Control at mRNA splicing (alternate splicing)(alternate splicing)

(four exons)

32 amino acidsReduces bone resorption

37 amino acidsVasodilator

bull1048766Messenger RNA longevity can be influenced by several factorsbull1048766Poly(A) tails seem to stabilize mRNAsbull1048766The sequence of the 3rsquountranslated region (3rsquoUTR) preceding a poly(A) tail also seems to affect mRNA stabilitybull1048766Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3rsquountranslated regions

bull1048766Messenger RNA longevity can be influenced by several factorsbull1048766Poly(A) tails seem to stabilize mRNAsbull1048766The sequence of the 3rsquountranslated region (3rsquoUTR) preceding a poly(A) tail also seems to affect mRNA stabilitybull1048766Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3rsquountranslated regions

6- Control at mRNA stability6- Control at mRNA stability

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

6- Control at mRNA stability6- Control at mRNA stability

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

6- Control at mRNA stability6- Control at mRNA stability

7- Control at initiation of translation7- Control at initiation of translation

5rsquo UTR

3rsquo UTRAUG UAA

Specific sequences make specific secondary structures

Specific protein factors bind to these secondary structures

COOH+NH2

NH2

ATP

CO NH

CO NH

ubiquitin protein ligase

Doomed protein

molecule

26S proteaso

me

bullUbiquitin-dependent proteolysis bullProtein molecule is tagged for degradation by attachment of a 20 kDa protein ubiquitin

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

8-Regulation by protein stability8-Regulation by protein stability

Similarity of regulation between eukaryotes and prokaryote

1Principles are the same signals (信号 ) activators and repressors (激活蛋白和阻遏蛋白 )recruitment and allostery cooperative binding (招募异构和协同结合 )

2 The gene expression steps subjected to regulation are similar and the initiation of transcription is the most pervasively regulated step

Difference in regulation between eukaryotes and prokaryote

1 Pre-mRNA splicing adds an important step for regulation (mRNA前体的剪接 )

2 The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart (真核转录机器更复杂 )

3 Nucleosomes and their modifiers influence access to genes (核小体及其修饰体 )

4 Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes (真核基因有更多结合位点 )

15

A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration

Bacteria

Yeast

Human

16

Promoter

Core promoter in eukaryote TATA-box Initiator (Inr) in prokaryote -10 region Inr

Proximal elements of promoter in prokaryote -35 region in eukaryote CAAT-box GC-box UPE upstream promoter element UAS upstream activating sequence Terminator ( 终止子 ) A DNA sequence just

downstream of the coding segment of a gene which is recognized by RNA polymerase as a signal to stop transcription

Cis-acting elementCis-acting element

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

4- Control at transcription 4- Control at transcription initiationinitiation

gene control region for gene X

By using different sequences (promoter enhancer or silencer sequences) and factors the rate of transcription of a gene is controlled

By using different sequences (promoter enhancer or silencer sequences) and factors the rate of transcription of a gene is controlled

Calcitonin gene-related peptide

61

5- Control at mRNA splicing 5- Control at mRNA splicing (alternate splicing)(alternate splicing)

(four exons)

32 amino acidsReduces bone resorption

37 amino acidsVasodilator

bull1048766Messenger RNA longevity can be influenced by several factorsbull1048766Poly(A) tails seem to stabilize mRNAsbull1048766The sequence of the 3rsquountranslated region (3rsquoUTR) preceding a poly(A) tail also seems to affect mRNA stabilitybull1048766Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3rsquountranslated regions

bull1048766Messenger RNA longevity can be influenced by several factorsbull1048766Poly(A) tails seem to stabilize mRNAsbull1048766The sequence of the 3rsquountranslated region (3rsquoUTR) preceding a poly(A) tail also seems to affect mRNA stabilitybull1048766Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3rsquountranslated regions

6- Control at mRNA stability6- Control at mRNA stability

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

6- Control at mRNA stability6- Control at mRNA stability

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

6- Control at mRNA stability6- Control at mRNA stability

7- Control at initiation of translation7- Control at initiation of translation

5rsquo UTR

3rsquo UTRAUG UAA

Specific sequences make specific secondary structures

Specific protein factors bind to these secondary structures

COOH+NH2

NH2

ATP

CO NH

CO NH

ubiquitin protein ligase

Doomed protein

molecule

26S proteaso

me

bullUbiquitin-dependent proteolysis bullProtein molecule is tagged for degradation by attachment of a 20 kDa protein ubiquitin

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

8-Regulation by protein stability8-Regulation by protein stability

Similarity of regulation between eukaryotes and prokaryote

1Principles are the same signals (信号 ) activators and repressors (激活蛋白和阻遏蛋白 )recruitment and allostery cooperative binding (招募异构和协同结合 )

2 The gene expression steps subjected to regulation are similar and the initiation of transcription is the most pervasively regulated step

Difference in regulation between eukaryotes and prokaryote

1 Pre-mRNA splicing adds an important step for regulation (mRNA前体的剪接 )

2 The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart (真核转录机器更复杂 )

3 Nucleosomes and their modifiers influence access to genes (核小体及其修饰体 )

4 Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes (真核基因有更多结合位点 )

15

A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration

Bacteria

Yeast

Human

16

Promoter

Core promoter in eukaryote TATA-box Initiator (Inr) in prokaryote -10 region Inr

Proximal elements of promoter in prokaryote -35 region in eukaryote CAAT-box GC-box UPE upstream promoter element UAS upstream activating sequence Terminator ( 终止子 ) A DNA sequence just

downstream of the coding segment of a gene which is recognized by RNA polymerase as a signal to stop transcription

Cis-acting elementCis-acting element

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Calcitonin gene-related peptide

61

5- Control at mRNA splicing 5- Control at mRNA splicing (alternate splicing)(alternate splicing)

(four exons)

32 amino acidsReduces bone resorption

37 amino acidsVasodilator

bull1048766Messenger RNA longevity can be influenced by several factorsbull1048766Poly(A) tails seem to stabilize mRNAsbull1048766The sequence of the 3rsquountranslated region (3rsquoUTR) preceding a poly(A) tail also seems to affect mRNA stabilitybull1048766Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3rsquountranslated regions

bull1048766Messenger RNA longevity can be influenced by several factorsbull1048766Poly(A) tails seem to stabilize mRNAsbull1048766The sequence of the 3rsquountranslated region (3rsquoUTR) preceding a poly(A) tail also seems to affect mRNA stabilitybull1048766Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3rsquountranslated regions

6- Control at mRNA stability6- Control at mRNA stability

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

6- Control at mRNA stability6- Control at mRNA stability

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

6- Control at mRNA stability6- Control at mRNA stability

7- Control at initiation of translation7- Control at initiation of translation

5rsquo UTR

3rsquo UTRAUG UAA

Specific sequences make specific secondary structures

Specific protein factors bind to these secondary structures

COOH+NH2

NH2

ATP

CO NH

CO NH

ubiquitin protein ligase

Doomed protein

molecule

26S proteaso

me

bullUbiquitin-dependent proteolysis bullProtein molecule is tagged for degradation by attachment of a 20 kDa protein ubiquitin

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

8-Regulation by protein stability8-Regulation by protein stability

Similarity of regulation between eukaryotes and prokaryote

1Principles are the same signals (信号 ) activators and repressors (激活蛋白和阻遏蛋白 )recruitment and allostery cooperative binding (招募异构和协同结合 )

2 The gene expression steps subjected to regulation are similar and the initiation of transcription is the most pervasively regulated step

Difference in regulation between eukaryotes and prokaryote

1 Pre-mRNA splicing adds an important step for regulation (mRNA前体的剪接 )

2 The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart (真核转录机器更复杂 )

3 Nucleosomes and their modifiers influence access to genes (核小体及其修饰体 )

4 Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes (真核基因有更多结合位点 )

15

A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration

Bacteria

Yeast

Human

16

Promoter

Core promoter in eukaryote TATA-box Initiator (Inr) in prokaryote -10 region Inr

Proximal elements of promoter in prokaryote -35 region in eukaryote CAAT-box GC-box UPE upstream promoter element UAS upstream activating sequence Terminator ( 终止子 ) A DNA sequence just

downstream of the coding segment of a gene which is recognized by RNA polymerase as a signal to stop transcription

Cis-acting elementCis-acting element

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

bull1048766Messenger RNA longevity can be influenced by several factorsbull1048766Poly(A) tails seem to stabilize mRNAsbull1048766The sequence of the 3rsquountranslated region (3rsquoUTR) preceding a poly(A) tail also seems to affect mRNA stabilitybull1048766Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3rsquountranslated regions

bull1048766Messenger RNA longevity can be influenced by several factorsbull1048766Poly(A) tails seem to stabilize mRNAsbull1048766The sequence of the 3rsquountranslated region (3rsquoUTR) preceding a poly(A) tail also seems to affect mRNA stabilitybull1048766Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3rsquountranslated regions

6- Control at mRNA stability6- Control at mRNA stability

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

6- Control at mRNA stability6- Control at mRNA stability

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

6- Control at mRNA stability6- Control at mRNA stability

7- Control at initiation of translation7- Control at initiation of translation

5rsquo UTR

3rsquo UTRAUG UAA

Specific sequences make specific secondary structures

Specific protein factors bind to these secondary structures

COOH+NH2

NH2

ATP

CO NH

CO NH

ubiquitin protein ligase

Doomed protein

molecule

26S proteaso

me

bullUbiquitin-dependent proteolysis bullProtein molecule is tagged for degradation by attachment of a 20 kDa protein ubiquitin

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

8-Regulation by protein stability8-Regulation by protein stability

Similarity of regulation between eukaryotes and prokaryote

1Principles are the same signals (信号 ) activators and repressors (激活蛋白和阻遏蛋白 )recruitment and allostery cooperative binding (招募异构和协同结合 )

2 The gene expression steps subjected to regulation are similar and the initiation of transcription is the most pervasively regulated step

Difference in regulation between eukaryotes and prokaryote

1 Pre-mRNA splicing adds an important step for regulation (mRNA前体的剪接 )

2 The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart (真核转录机器更复杂 )

3 Nucleosomes and their modifiers influence access to genes (核小体及其修饰体 )

4 Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes (真核基因有更多结合位点 )

15

A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration

Bacteria

Yeast

Human

16

Promoter

Core promoter in eukaryote TATA-box Initiator (Inr) in prokaryote -10 region Inr

Proximal elements of promoter in prokaryote -35 region in eukaryote CAAT-box GC-box UPE upstream promoter element UAS upstream activating sequence Terminator ( 终止子 ) A DNA sequence just

downstream of the coding segment of a gene which is recognized by RNA polymerase as a signal to stop transcription

Cis-acting elementCis-acting element

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

bull1048766When this sequence is artificially transferred to the 3rsquountranslated region of more stable mRNAs they too become unstablebull1048766Chemical factors such as hormones may also affect mRNA stablilitybull1048766In the toad Xenopus laevis( 非洲爪蟾 ) the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激素 ) However in addition to inducing transcription of this gene estrogen also increases the longevity of its mRNA

6- Control at mRNA stability6- Control at mRNA stability

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

6- Control at mRNA stability6- Control at mRNA stability

7- Control at initiation of translation7- Control at initiation of translation

5rsquo UTR

3rsquo UTRAUG UAA

Specific sequences make specific secondary structures

Specific protein factors bind to these secondary structures

COOH+NH2

NH2

ATP

CO NH

CO NH

ubiquitin protein ligase

Doomed protein

molecule

26S proteaso

me

bullUbiquitin-dependent proteolysis bullProtein molecule is tagged for degradation by attachment of a 20 kDa protein ubiquitin

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

8-Regulation by protein stability8-Regulation by protein stability

Similarity of regulation between eukaryotes and prokaryote

1Principles are the same signals (信号 ) activators and repressors (激活蛋白和阻遏蛋白 )recruitment and allostery cooperative binding (招募异构和协同结合 )

2 The gene expression steps subjected to regulation are similar and the initiation of transcription is the most pervasively regulated step

Difference in regulation between eukaryotes and prokaryote

1 Pre-mRNA splicing adds an important step for regulation (mRNA前体的剪接 )

2 The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart (真核转录机器更复杂 )

3 Nucleosomes and their modifiers influence access to genes (核小体及其修饰体 )

4 Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes (真核基因有更多结合位点 )

15

A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration

Bacteria

Yeast

Human

16

Promoter

Core promoter in eukaryote TATA-box Initiator (Inr) in prokaryote -10 region Inr

Proximal elements of promoter in prokaryote -35 region in eukaryote CAAT-box GC-box UPE upstream promoter element UAS upstream activating sequence Terminator ( 终止子 ) A DNA sequence just

downstream of the coding segment of a gene which is recognized by RNA polymerase as a signal to stop transcription

Cis-acting elementCis-acting element

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

bull Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small noncoding RNA molecules called microRNA (miRNAs)

6- Control at mRNA stability6- Control at mRNA stability

7- Control at initiation of translation7- Control at initiation of translation

5rsquo UTR

3rsquo UTRAUG UAA

Specific sequences make specific secondary structures

Specific protein factors bind to these secondary structures

COOH+NH2

NH2

ATP

CO NH

CO NH

ubiquitin protein ligase

Doomed protein

molecule

26S proteaso

me

bullUbiquitin-dependent proteolysis bullProtein molecule is tagged for degradation by attachment of a 20 kDa protein ubiquitin

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

8-Regulation by protein stability8-Regulation by protein stability

Similarity of regulation between eukaryotes and prokaryote

1Principles are the same signals (信号 ) activators and repressors (激活蛋白和阻遏蛋白 )recruitment and allostery cooperative binding (招募异构和协同结合 )

2 The gene expression steps subjected to regulation are similar and the initiation of transcription is the most pervasively regulated step

Difference in regulation between eukaryotes and prokaryote

1 Pre-mRNA splicing adds an important step for regulation (mRNA前体的剪接 )

2 The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart (真核转录机器更复杂 )

3 Nucleosomes and their modifiers influence access to genes (核小体及其修饰体 )

4 Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes (真核基因有更多结合位点 )

15

A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration

Bacteria

Yeast

Human

16

Promoter

Core promoter in eukaryote TATA-box Initiator (Inr) in prokaryote -10 region Inr

Proximal elements of promoter in prokaryote -35 region in eukaryote CAAT-box GC-box UPE upstream promoter element UAS upstream activating sequence Terminator ( 终止子 ) A DNA sequence just

downstream of the coding segment of a gene which is recognized by RNA polymerase as a signal to stop transcription

Cis-acting elementCis-acting element

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

7- Control at initiation of translation7- Control at initiation of translation

5rsquo UTR

3rsquo UTRAUG UAA

Specific sequences make specific secondary structures

Specific protein factors bind to these secondary structures

COOH+NH2

NH2

ATP

CO NH

CO NH

ubiquitin protein ligase

Doomed protein

molecule

26S proteaso

me

bullUbiquitin-dependent proteolysis bullProtein molecule is tagged for degradation by attachment of a 20 kDa protein ubiquitin

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

8-Regulation by protein stability8-Regulation by protein stability

Similarity of regulation between eukaryotes and prokaryote

1Principles are the same signals (信号 ) activators and repressors (激活蛋白和阻遏蛋白 )recruitment and allostery cooperative binding (招募异构和协同结合 )

2 The gene expression steps subjected to regulation are similar and the initiation of transcription is the most pervasively regulated step

Difference in regulation between eukaryotes and prokaryote

1 Pre-mRNA splicing adds an important step for regulation (mRNA前体的剪接 )

2 The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart (真核转录机器更复杂 )

3 Nucleosomes and their modifiers influence access to genes (核小体及其修饰体 )

4 Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes (真核基因有更多结合位点 )

15

A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration

Bacteria

Yeast

Human

16

Promoter

Core promoter in eukaryote TATA-box Initiator (Inr) in prokaryote -10 region Inr

Proximal elements of promoter in prokaryote -35 region in eukaryote CAAT-box GC-box UPE upstream promoter element UAS upstream activating sequence Terminator ( 终止子 ) A DNA sequence just

downstream of the coding segment of a gene which is recognized by RNA polymerase as a signal to stop transcription

Cis-acting elementCis-acting element

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

COOH+NH2

NH2

ATP

CO NH

CO NH

ubiquitin protein ligase

Doomed protein

molecule

26S proteaso

me

bullUbiquitin-dependent proteolysis bullProtein molecule is tagged for degradation by attachment of a 20 kDa protein ubiquitin

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛素激活酶 (E1) 泛素交联酶 (E2) 泛素连接酶 (E3) 和 26S 蛋白酶体组成 是降解细胞内蛋白质的主要途径

对于许多细胞进程包括细胞周期基因表达的调控氧化应激反应等都是必不可少的 2004 年诺贝尔化学奖

8-Regulation by protein stability8-Regulation by protein stability

Similarity of regulation between eukaryotes and prokaryote

1Principles are the same signals (信号 ) activators and repressors (激活蛋白和阻遏蛋白 )recruitment and allostery cooperative binding (招募异构和协同结合 )

2 The gene expression steps subjected to regulation are similar and the initiation of transcription is the most pervasively regulated step

Difference in regulation between eukaryotes and prokaryote

1 Pre-mRNA splicing adds an important step for regulation (mRNA前体的剪接 )

2 The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart (真核转录机器更复杂 )

3 Nucleosomes and their modifiers influence access to genes (核小体及其修饰体 )

4 Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes (真核基因有更多结合位点 )

15

A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration

Bacteria

Yeast

Human

16

Promoter

Core promoter in eukaryote TATA-box Initiator (Inr) in prokaryote -10 region Inr

Proximal elements of promoter in prokaryote -35 region in eukaryote CAAT-box GC-box UPE upstream promoter element UAS upstream activating sequence Terminator ( 终止子 ) A DNA sequence just

downstream of the coding segment of a gene which is recognized by RNA polymerase as a signal to stop transcription

Cis-acting elementCis-acting element

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Similarity of regulation between eukaryotes and prokaryote

1Principles are the same signals (信号 ) activators and repressors (激活蛋白和阻遏蛋白 )recruitment and allostery cooperative binding (招募异构和协同结合 )

2 The gene expression steps subjected to regulation are similar and the initiation of transcription is the most pervasively regulated step

Difference in regulation between eukaryotes and prokaryote

1 Pre-mRNA splicing adds an important step for regulation (mRNA前体的剪接 )

2 The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart (真核转录机器更复杂 )

3 Nucleosomes and their modifiers influence access to genes (核小体及其修饰体 )

4 Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes (真核基因有更多结合位点 )

15

A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration

Bacteria

Yeast

Human

16

Promoter

Core promoter in eukaryote TATA-box Initiator (Inr) in prokaryote -10 region Inr

Proximal elements of promoter in prokaryote -35 region in eukaryote CAAT-box GC-box UPE upstream promoter element UAS upstream activating sequence Terminator ( 终止子 ) A DNA sequence just

downstream of the coding segment of a gene which is recognized by RNA polymerase as a signal to stop transcription

Cis-acting elementCis-acting element

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Difference in regulation between eukaryotes and prokaryote

1 Pre-mRNA splicing adds an important step for regulation (mRNA前体的剪接 )

2 The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart (真核转录机器更复杂 )

3 Nucleosomes and their modifiers influence access to genes (核小体及其修饰体 )

4 Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes (真核基因有更多结合位点 )

15

A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration

Bacteria

Yeast

Human

16

Promoter

Core promoter in eukaryote TATA-box Initiator (Inr) in prokaryote -10 region Inr

Proximal elements of promoter in prokaryote -35 region in eukaryote CAAT-box GC-box UPE upstream promoter element UAS upstream activating sequence Terminator ( 终止子 ) A DNA sequence just

downstream of the coding segment of a gene which is recognized by RNA polymerase as a signal to stop transcription

Cis-acting elementCis-acting element

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

15

A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration

Bacteria

Yeast

Human

16

Promoter

Core promoter in eukaryote TATA-box Initiator (Inr) in prokaryote -10 region Inr

Proximal elements of promoter in prokaryote -35 region in eukaryote CAAT-box GC-box UPE upstream promoter element UAS upstream activating sequence Terminator ( 终止子 ) A DNA sequence just

downstream of the coding segment of a gene which is recognized by RNA polymerase as a signal to stop transcription

Cis-acting elementCis-acting element

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

16

Promoter

Core promoter in eukaryote TATA-box Initiator (Inr) in prokaryote -10 region Inr

Proximal elements of promoter in prokaryote -35 region in eukaryote CAAT-box GC-box UPE upstream promoter element UAS upstream activating sequence Terminator ( 终止子 ) A DNA sequence just

downstream of the coding segment of a gene which is recognized by RNA polymerase as a signal to stop transcription

Cis-acting elementCis-acting element

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Enhancer ( 激活元件 ) a given site binds regulator responsible for activating the gene Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals Activation at a distance is much more common in eukaryotes

Silencer （沉默子） A DNA sequence that helps to reduce or shut off the expression of a nearby gene

Insulators ( 绝缘子 ) or boundary elements ( 临界元件 ) are regulatory sequences between enhancers and promoters They block activation of a linked promoter by activator bound at the enhancer and therefore ensure activators work discriminately

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

What is trans-acting factor

Usually they are proteins that bind

to the cis-acting elements to control

gene expression

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

These trans-acting factors can control

gene expression in several ways may be expressed in a specific tissue

may be expressed at specific time in

development

may be required for protein modification

may be activated by ligand binding

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

(1) RNA polymerase

prokaryotic RNA Pol

eukaryotic RNA Pol

(2) Transcription factors

Basalgeneral TFs

Specific TFs

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

(3) Domains of trans-acting factors

DNA binding domain DBD

DNA结合结构域 transcription activating domain

转录活化结构域

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

Topic 1 Conserved Topic 1 Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

一真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

25

The basic features of gene regulation are the same in all eukaryotes because of the similarity in their transcription and nucleosome structureYeast is the most amenable to both genetic and biochemical dissection and produces much of knowledge of the action of the eukaryotic repressor and activator The typical eukaryotic activators works in a manner similar to the simplest bacterial caseRepressors work in a variety of ways

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

1 Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions which are very often on separate domains of the protein

Gal4 bound to its site on DNA

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

The regulatory sequences of the Yeast GAL1 gene

Eukaryotic activators---Example 1 Gal4 Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the

yeast S cerevisae Gal4 binds to four sites (UASG) upstream of GAL1(5-

CGGRNNRCYNYNYNCNCCG-3 ) and activates transcription 1000-fold in the presence of galactose

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Experimental evidences showing that Gal4 contains separate DNA binding and activating domains

1 Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription

2 Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor LexA activates the transcription of the reporter gene Domain swap experimentDomain swap experiment

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

29

Domain swap experiment Moving domains among proteins proving that domains can be dissected into separate parts of the proteins

Many similar experiments shows that DNA binding domains and activating regions are separable

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

30

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4 respectively

Produce fusion proteins

Box1 The two hybrid Assay (two hybrid Assay ( 双杂交双杂交 )) to study protein-protein interaction and identify proteins interacting with a known protein in cells

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

2 Eukaryotic regulators use a range of DNA binding domains but DNA recognition involves the same principles as found in bacteria

Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger （锌指） and zinc cluster Leucine zipper motif （亮氨酸拉链） Helix-Loop-Helix proteins （螺旋 - 突环 - 螺旋） basic

zipper and HLH proteins

Transcription factor motifs

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

HTH (helix-turn-

helix)α-helix (N-terminus)----specific

α-helix (C-terminus)----non-specific

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Bacterial regulatory proteinsbull Most use the helix-turn-helix (HTH 旋转 - 转角 - 旋

转） motif to bind DNA targetbull Most bind as dimers to DNA sequence each

monomer inserts an a helix into the major groove

Eukaryotic regulatory proteins1 Recognize the DNA using the similar principles

with some variations in detail2 In addition to form homodimers ( 同源二聚体 )

some form heterodimers ( 异源二聚体 ) to recognize DNA extending the range of DNA-binding specificity

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Zinc containing DNA-binding domains ( 锌指结构域 ) Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Leucine Zipper Motif ( 亮氨酸拉链基序 ) The Motif combines dimerization and DNA-binding surfaces within a single structural unit

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Helix-Loop-Helix motif

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

myogenic factor 生肌调节蛋白是一种转录因子

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

3 Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content

Acidic activation region ( 酸性激活区域 ) contain both critical acidic amino acids and hydrophobic aa yeast Gal4yeast Gal4

Glutamine-rich region ( 谷氨酰胺富集区 ) mammalian activator SP1

Proline-rich region ( 脯氨酸富集区 ) mammalian activator CTF1

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

Topic 2 Recruitment of Topic 2 Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

二真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment amp Activation at a distance

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Eukaryotic activators ( 真核激活蛋白 ) also work by recruiting ( 招募 ) as in bacteria but recruit polymerase indirectly in two ways

1 Interacting with parts of the transcription machinery2 Recruiting nucleosome

modifiers that alter chromatin in the vicinity of a gene

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

The eukaryotic transcriptional machineryeukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes such as the Mediator and the TFⅡD complex Activators interact with one or more of these complexes and recruit them to the gene

1 Activators recruit the transcription machinery to the gene

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Chromatin Immuno-precipitation (ChIP) (Chromatin Immuno-precipitation (ChIP) ( 染色染色质免疫共沉淀质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

2 Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome

modifiers Those add chemical groups to the tails of histones ( 在组蛋白尾上加化学基团 ) such as histone acetyl transferases (HATs)Those remodel the nucleosomes ( 重塑核小体 ) such as the ATP-dependent activity of SWISNF

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

How the nucleosome modification help activate a gene

1 ldquoLoosenrdquo the chromatin structure by chromosome remodeling and histone modification such as acetylation which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Local alterations in chromatin structure directed by activatorsActivators capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessiblewithin chromain

(a) The activator is shown recruiting a histone acetylase That enzyme adds acetyl groups to residues withinthe histone tails This alters the packing of the nucleosomes somewhat and also creates binding sites for proteins carrying the appropriate recognition domains

(b)The activator recruits a nucleosome remodeller which alters the structure of nucleosomes around the promoter rendering it accessible and capable of binding the transcription machinery

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators preventing the activating the non-specific genes

3 Action at a distance loops and insulators

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Insulators block activation by enhancers

a) A promoter activated by activators bound to an enhancerb) An insulator is placed between the enhancer and the promoter When bound by appropriate insulator- binding proteins activation of the promoter by the enhancer is blocked despite activators binding to the

enhancerc) The activator can activate another promoter nearbyd) The original promoter can be activated by another enhancer placed downstream

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Transcriptional Silencing Transcriptional Silencing (( 转录沉转录沉默默 ))

Transcriptional Silencing is a specialized form of repression that can spread along chromatin switching off multiple genes without the need for each to bear binding sites for specific repressor

Insulator elements can block this spreading so insulators protect genes from both indiscriminate activation and repression

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

4 Appropriate regulation of some groups of genes requires locus control region (LCR)

1 Human and mouse globin genes are clustered in genome and differently expressed at different stages of development

2 A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR) is found 30-50 kb upstream of the cluster of globin genes It binds regulatory proteins that cause the chromatin structure to ldquoopen uprdquo allowing access to the array of regulators that control expression of the individual genes in a defined order

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Regulation by LCRs

(a)The human globin genes and the LCR that ensures their ordered expression(b) The globin genes from mice which are also regulated by an LCR(C) The HoxD gene cluster from the mouse controlled by an element called the GCR which likethe LCRs appears to impose ordered expression on the gene cluster

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Topic 3 TranscriptionalTopic 3 TranscriptionalRepressor amp its regulationRepressor amp its regulationTopic 3 TranscriptionalTopic 3 Transcriptional

Repressor amp its regulationRepressor amp its regulation

In eukaryotes most repressors do do notnot repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase (Bacteria often do so)

三真核转录阻遏蛋白（或抑制蛋白）及其调控三真核转录阻遏蛋白（或抑制蛋白）及其调控

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Commonly eukaryotic repressors recruit nucleosome modifiersrecruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups] Some modifier adds methyl groups to the histone tails which frequently repress the transcription

This modification causes transcriptional silencing

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Three other ways in which an eukaryotic repressor works include

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site

(2)Binds to a site different from that of the activator but physically physically interacts with an activatorinteracts with an activator and thus block its activating region

(3)Binds to a site upstream of the promoter physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Ways in which eukaryotic repressor workWays in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Binds to the transcription machineryRecruits nucleosome

modifiers (most common)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

In the presence of glucose Mig1 binds to a site between the USAG and the GAL1 promoter and recruits the Tup1 repressing complex Tup1 recruits recruits histone deacetylaseshistone deacetylases and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription

A specific example Repression of the GAL1 gene in yeast

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

Topic 4 Signal Topic 4 Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

四基于真核转录调控的前沿学科信号传导Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

1 Signals are often communicatedcommunicated to transcriptional regulators through signal transduction pathway信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Environmental SignalsInformationEnvironmental SignalsInformation ( 信号 )

1 Small molecules such as sugar histamine ( 组胺 )

2 Proteins released by one cell and received by another

In eukaryotic cells most signals are most signals are communicated to genes through communicated to genes through signal transduction pathway signal transduction pathway (indirect)(indirect) in which the initiating ligand is detected by a specific cell surface receptor

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

3 The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulatortranscriptional regulator

Signal transduction pathway

1 The initial ligand (ldquosignalrdquo) binds to an extracellular domainextracellular domain of a specific cell surface receptor

2 The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4 The transcriptional regulator control the target gene target gene expressionexpression

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity allowing trans-phosphorylation

The activated JAKs subsequently phosphorylate additional targets including both the receptors and the major substrates STATs

STATs are latent transcription factors that reside in the cytoplasm until activated

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

MAP kinases are activated within the protein kinase cascades called ldquoMAPK cascaderdquo

Each one consists of three enzymes MAP kinase MAP kinase kinase (MKK MEK or MAP2K) and MAP kinase kinase kinase (MKKK MEKK or MAP3K) that are activated in series

A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

信号通过不同方式控制转录调节蛋白的活性

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Mechanism 1 unmasking an activating Mechanism 1 unmasking an activating regionregion

(1) A conformational change to reveal the previously buried activating region

(2) Releasing of the previously bound masking protein Example the activator Gal4 is controlled by the masking Gal80)

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit a deacetylase enzyme to repress the target genes Example Rb represses the function of the mammalian transcription activator E2F in this way Phosphorylation of Rb releases E2F to activate the target gene expression

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Mechanism 2 Transport into and out of Mechanism 2 Transport into and out of the nucleusthe nucleusWhen not active many activators and repressors are held in the cytoplasm The signaling ligand causes them to move into the nucleus where they activate transcription

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Other Mechanisms 1 Other Mechanisms 1 A cascade of A cascade of kinases that ultimately causekinases that ultimately cause the the phosphorylation of regulator phosphorylation of regulator in in nucleusnucleus (new(new) (Fig19-4a)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)

Other Mechanisms 2 Other Mechanisms 2 The activated The activated receptor is cleaved by cellular receptor is cleaved by cellular proteases proteases (( 蛋白酶蛋白酶 )) and and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the enters the nuclease and activates the regulatornuclease and activates the regulator (new(new)(Fig19-4c)