6-11-13 Cell Bio Lecture 7BB (1)

7/28/2019 6-11-13 Cell Bio Lecture 7BB (1)

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Lecture 7Control of Gene Expression

Reading: Chapter 7



Figure 7-1 Molecular Biology of the Cell (© Garland Science 2008)

The different cell types in a multicellular organism

differ dramatically in both structure and function.



Figure 7-2a Molecular Biology of the Cell (© Garland Science 2008)

Evidence for the preservation of the

genome during cell differentiation

•The injected donor nucleus is capable of programming the

recipient egg to produce a normal tadpole



Figure 7-2b Molecular Biology of the Cell (© Garland Science 2008)



• First differentiated pieces of plant tissue are placed in

culture and then dissociated into single cells.

• Often, one of these individual cells can regenerate an entire

adult plant



Figure 7-2c Molecular Biology of the Cell (© Garland Science 2008)





Different Cell Types Synthesize Different Sets of

Proteins

1. Many processes are common to all cells, and any two

cells in a single organism therefore have many proteins

in common

2. Some proteins are abundant in the specialized cells inwhich they function and cannot be detected

elsewhere, even by sensitive tests. Hemoglobin, for

example, can be detected only in red blood cells.

3. At any one time, a human cell expresses 30-60% of its

approximately 25,000 genes


http://slidepdf.com/reader/full/6-11-13-cell-bio-lecture-7bb-1 7/88Figure 7-3 Molecular Biology of the Cell (© Garland Science 2008)

Differences in mRNA expression

patterns among different types of human cancer cells

Microarray

• When the patterns of mRNAs in a series of different human cell lines are

compared, it is found that the level of expression of almost every active

gene varies from one cell type to another.



Different Cell Types Synthesize Different Sets of Proteins

1. Many processes are common to all cells, and any two

cells in a single organism therefore have many proteins

in common

2. Some proteins are abundant in the specialized cells in

which they function and cannot be detected

elsewhere, even by sensitive tests. Hemoglobin, for

example, can be detected only in red blood cells.

3. At any one time, a human cell expresses 30-60% of its

approximately 25,000 genes4. There are many steps after transcription at which gene

expression can be regulated



Regulation of Gene Expression

• Gene expression is regulated at many of the steps in the

pathway from DNA to RNA to protein

1. Transcriptional control

2. RNA processing control3. RNA transport and localization control

4. Translational control

5. mRNA degradation control

6. Protein activity control



Steps at which eukaryotic gene

expression can be controlled



Cells can change the pattern of genes they

express in response to changes in their environment,such as signals from other cells



How does a cell determine which of itsthousands of genes to transcribe?

• The transcription of each gene is controlled by a

regulatory region of DNA relatively near the site where

transcription begins• Some regulatory regions are simple and act as

switches thrown by a single signal

• Many others are complex: respond to a variety of

signals to turn neighboring gene on or off



How does a cell determine which of itsthousands of genes to transcribe?

Two components:

(1) short stretches of DNA of defined sequence

(2) gene regulatory proteins that recognize andbind to this DNA

Whether complex or simple, these switching devices are

found in all cells and are composed of two types of

fundamental components:



gene

regulatory

proteins

The Outside of the DNA Helix Can Be Read by protein

Gene regulatory proteins recognizesequences specifically by binding to

the outside of the DNA

-specifically at ______ ?



The Outside of the DNA Helix Can Be Read by protein

In the major groove the

patterns are markedly

different for each of the four

base-pair arrangements


http://slidepdf.com/reader/full/6-11-13-cell-bio-lecture-7bb-1 16/88Table 7-1 Molecular Biology of the Cell (© Garland Science 2008)



Gene Regulatory Proteins Contain Structural

Motifs That Can Read DNA Sequences

• Molecular recognition in biology generally relies on

an exact fit between the surfaces of two molecules




•Although each individual contact is weak, the 20 or so

that are typically formed at the protein-DNA interface

add together to ensure that the interaction is both highly

specific and very strong

• Protein makes a series

of contacts with the

DNA, involving hydrogen

bonds, ionic bonds, and

hydrophobicinteractions.



Gene regulatory proteins recognize specific

sequences of DNA

• Gene regulatory proteins recognize DNAthrough small number of structural motifs.Some common motifs,

- helix turn helix

- homeodomain

- zinc finger

- Leucine zipper

• Specific amino acid sequence in the motif is

important for specificity of binding to DNA

• These proteins mostly bind as homodimersor heterodimers



DNA Binding Motifs

Helix-turn-helix• Constructed from two α helices connected by a short

extended chain of amino acids, which constitutes the "turn”

helix fits into the major

groove of DNA

• The C-terminal helix is called

the recognition helix: (?)

•The N-terminal α helix(?)



Some helix-turn-helix DNA-binding proteins

All of the proteins bind DNA as dimers

DNA binding motifs



Homeodomains

• Are special class of Helix-Loop-Helix proteins• also contain a helix-loop-helix which is presented almost

identically in all homeodomain proteins

• these proteins were classified as homeodomain because theyhad a almost identical 60 aa stretch

DNA binding motifs



Zinc fingers

• DNA binding motifs of these contain Zn atoms coordinated

• Usually form dimers, one of the two α- helices of each subunit

interact with major groove of DNA

DNA binding motifs




Some Proteins Use Loops That Enter the Major and

Minor Grooves to Recognize DNA

• These proteins use protruding

peptide loops to read

nucleotide sequences, ratherthan α and ß sheets

e.g. p53



Leucine zipper

• helices of two monomers joined together to form a short

coiled-coil formed by interactions of hydrophobic amino acids

(usually Leu)

DNA binding motifs



The control of gene expression is governed

primarily by DNA-binding proteins that

recognize specific control sequences of genes



• Gene regulation has been studied extensively in

E. coli

• Highly efficient genetic mechanisms have evolvedto turn transcription of specific genes on and off

depending on a cell's metabolic need for specificgene products

• These responses can be due to changes in theenvironment as well as non environmentallyregulated cellular activity and cell division



• Bacteria adapt to their environment by producing

certain enzymes ( _________ enzymes) only when

specific substrates are present

• Enzymes continuously produced regardless of

chemical makeup of the environment are called _____________ enzymes

•

An abundance of an end product in the environmentrepresses gene expression

– Repressible system



• Regulation of the inducible or repressible type

may be under positive control or negativecontrol

– Negative control:

– Positive control:



The basics of prokaryotic transcriptionalregulation: Genetic switches



How Genetic Switches Work

• Basic components of genetic switches:

gene regulatory proteins and the specific DNA

sequences that these proteins recognize



Promoter: is a specific DNA sequence that directs RNA polymerase

to bind to DNA, to open the DNA double helix, and to begin

synthesizing an RNA molecule.

operator: is a short region of regulatory DNA of defined

nucleotide sequence that is recognized by a repressor protein

Operon: In a bacterial chromosome, a group of contiguous genes

that are transcribed into a single mRNA molecule




Tryptophan Operon




Tryptophan Operon

• The clustered genes in E. coli that code for enzymes that

manufacture the amino acid tryptophan.

• Five genes (A, B, C, D and E) are transcribed as a single

mRNA molecule.

• These genes are arranged as a single operon; they are

adjacent to one another on the chromosome and are

transcribed from a single promoter as one long mRNA

molecule

• The expression of many genes is regulated according to theavailable food in the environment




The Tryptophan Repressor Is a Simple Switch That Turns Genes

On and Off in Bacteria

Switching the Tryptophan Genes On and Off




The binding of tryptophan to the

tryptophan repressor protein changes its

conformation



• Because the active, DNA-binding form of the protein

serves to turn genes off, this mode of gene regulation

is called negative control

• The gene regulatory proteins that function in this

way are called transcriptional repressors or gene

repressor proteins.



Transcriptional Activators Turns Genes ON

• Many bacterial promoters are only marginally

functional on their own

• Poorly functioning promoters can be rescued by

gene regulatory proteins: Activators

• Positive regulation: bound activator protein

promotes transcription (initiation up to 1000-fold)



Lac Operon

The Lac operon in E. coli is under both:Negative :Positive :

• The Lac operon codes for proteins required totransport the disaccharide lactose into the cell and to

break it down

Lac operon is under dual control:A Transcriptional Activator and Transcriptional

Repressor Control



A Transcriptional Activator and a Transcriptional

Repressor Control (dual control) the Lac Operon

The Lac operon in E. coli is under both:Negative : Lac repressorPositive : CAP (Catabolite Activator Protein)

Activates genes that enable E. coli to use alternative carbon

sources when glucose is unavailable.

Low glucose levels Increase cAMP (intracellular signaling molecule)

Binds CAP

CAP-cAMP

bind to its specific DNA sequence near target promoters

and thereby turn on the appropriate genes




Lac Z gene




• Lactose addition increases the concentration of allolactose,

an isomer of lactose, which binds to the repressor protein and

removes it from the DNA

• Glucose addition decreases the concentration of cyclic AMP;

because cyclic AMP no longer binds to CAP, this gene activator

protein dissociates from the DNA, turning off the operon.










DNA L i O D i B t i l G



DNA Looping Occurs During Bacterial Gene

Regulation

• Lac repressor is a single tetrameric molecule

• It can bind two operators simultaneously, looping out the intervening DNA.

• The ability to bind simultaneously to two operators strengthens the overall

interaction of the Lac repressor with DNA and thereby leads to greater levels

of repression in the cell.



Eukaryotic Gene Regulation



Prokaryotes Eukaryotes

Structure of genome

Size of genome

Location of gene

transcription andtranslation

Gene clustering

Default state

of transcription

DNA structure

Eukaryotic Gene Control Region Consists of a Promoter



Eukaryotic Gene Control Region Consists of a Promoter

Plus Regulatory DNA Sequences

• Mediator and the general transcription factors are the same for all polymerase II

transcribed genes

• The gene regulatory proteins and the locations of their binding sites relative to the

promoter differ for each gene

Many gene regulatory

proteins also influence the

chromatin structure of the

DNA control region thereby

affecting transcription

initiation indirectly (notshown)



• A central component of gene regulation in eukaryotes is

Mediator , a 24- subunit complex, which serves as an

intermediary between gene regulatory proteins and RNA

polymerase.

• Mediator provides an extended contact area for gene

regulatory proteins compared to that provided by RNA

polymerase alone, as in bacteria

• The packaging of eukaryotic DNA into chromatin provides

many opportunities for transcriptional regulation not

available to bacteria

Eukaryotic Gene Regulation



Gene regulation in eukaryotes is more

complex than it is in prokaryotes

• In eukaryotes most genes are not found in operons

• The proteins and DNA sequences participating ineukaryotic gene regulation are more numerous

• Often many DNA-binding proteins act on a single switch,

with many separate switches per gene, and the regulatorysequences of these switches are often located far from

promoters



•

Access to eukaryotic gene promoters is restricted bychromatin

• Gene regulation in eukaryotes requires the activity of

large protein complexes that promote or restrict access to

gene promoters by RNA polymerase



• The DNA sites to which eukaryotic gene activatorproteins bind were originally called enhancers because

their presence "enhanced" the rate of transcription

initiation

• Activator proteins could be bound tens of thousands

of nucleotide pairs away from the promoter- DNA

looping



• Gene activator proteins have a modular design

consisting of two distinct domains:

1. DNA-binding: recognizes a specific DNA sequence2. Activation domain: domain-accelerates the rate of

transcription initiation

The Modular Structure of a Gene




The Modular Structure of a Gene

Activator Protein

Experiment showed the presence of independent DNA-binding

and transcription-activating domains in the yeast gene activator

protein Gal4

Gal4 is normally responsible for

activating the transcription of

yeast genes that code for the

enzymes that convert galactose

to glucose.

A chimeric gene regulatory

protein,requires a LexA

recognition sequence to

activate transcription.



F k ti ti t t i di t




Four ways eukaryotic activator proteins can direct

local alterations in chromatin structure to stimulate

transcription initiation

Four of the most important ways of locally altering chromatin are through: covalent

histone modifications, nucleosome remodeling, nucleosome removal, and

nucleosome replacement

i i l S (?)




Transcriptional Synergy (?)

• Compares the rate of transcription produced by threeexperimentally constructed regulatory regions in a

eukaryotic cell.

Additive effect of multiple activators

k i i hibi



Eukaryotic Gene Repressor Proteins Can Inhibit

Transcription in Various Ways

• Like bacteria, eukaryotes use gene repressor proteins

in addition to activator proteins to regulate transcription

of their genes

• Most eukaryotic repressors must work on a gene-by-gene basis

• Like gene activator proteins, many eukaryotic

repressor proteins act through more than one

mechanism at a given target gene, thereby ensuring

robust and efficient repression




A i di id l l t t i ft




• An individual gene regulatory protein can often

participate in more than one type of regulatory complex

• A protein might function, for example, in one case as part of a complex

that activates transcription and in another case as part of a complex that

represses transcription

• Red and the green proteins are shared by both activating and repressing

complexes



• Thus individual eukaryotic gene regulatory proteins

are not necessarily dedicated activators or repressors;

instead, they function as regulatory parts that are used

to build complexes whose function depends on the

final assembly of all of the individual components.



lnsulators Are DNA Sequences That PreventEucaryotic Gene Regulatory Proteins from

Influencing Distant Genes

• All genes have control regions, which dictate at

which times, under what conditions, and in what

tissues the gene will be expressed.

• We have also seen that eukaryotic gene regulatory

proteins can act across very long stretches of DNA.

How are control regions of different genes

kept from interfering with one another?

S h i di i i h i f




Schematic diagram summarizing the properties of

insulators and barrier sequences

Insulators directionally block the action of enhancers, and

barrier sequences prevent the spread of heterochromatin

Model for how enhancer-blocking insulators



Model for how enhancer-blocking insulators

might work

Insulators act by moving a promoter into a new loop, where it is

shielded from the enhancer

Some mechanisms of barrier action




Some mechanisms of barrier action

The tethering of a region of chromatin

to a large fixed site (e.g. nuclear pore

complex), can form a barrier that stops

the spread of heterochromatin

The tight binding of barrier

proteins to a group of nucleosomes

can compete with heterochromatin

spreading.

Barriers can erase the histone

marks that are required for

heterochromatin to spread by

recruiting a group of highly active

histone-modifying enzymes

Multicomponent genetic switch controlling the



Multicomponent genetic switch controlling the

transcription of the Drosophila Even-skipped

(Eve)• Plays an important part in the development of the Drosophila

embryo.

• If this gene is inactivated by mutation, many parts of the

embryo fail to form, and the embryo dies early in development

• At the stage of development when Eve begins to be expressed,the embryo is a single giant cell containing multiple nuclei in acommon cytoplasm: contains a mixture of gene regulatoryproteins that are distributed unevenly along the length of theembryo)

Provide positional information

The nonuniform distribution of four gene regulatory




The nonuniform distribution of four gene regulatory

proteins in an early Drosophila embryo

• Although the nuclei are initially identical, they rapidly begin to

express different genes because they are exposed to different

gene regulatory proteins.

At this stage the embryo is a syncytium, withmultiple nuclei in a common cytoplasm

The seven stripes of the protein encoded by the Even




The seven stripes of the protein encoded by the Even-

skipped (Eve) gene in a developing Drosophila embryo

•Two and one-half hours after fertilization, the egg was fixed and stained with

antibodies that recognize the Eve protein (green) and antibodies that recognize

the Giant protein (red)

Experiment demonstrating the modular




Experiment demonstrating the modular

construction of the Eve gene regulatory region

Experiment demonstrating the modular construction of



Figure 7-55b,c Molecular Biology of the Cell (© Garland Science 2008)

Experiment demonstrating the modular construction of

the Eve gene regulatory region.

When this artificial construct was reintroduced into the genome

of Drosophila embryos, the embryos expressed β-galactosidase (detectable by

histochemical staining) precisely in the position of the second of the seven Eve

stripes




Distribution of the gene regulatory proteins responsible

for ensuring that Eve is expressed in stripe 2



The Pattern of DNA Methylation

(Epigenetic Marker ?)

• In vertebrate cells, the methylation of cytosine

provides a powerful mechanism through which gene

expression patterns are passed on to progeny cells.• DNA methylation in vertebrate DNA is restricted to C

nucleotides in the sequence CG

• An enzyme called maintenance methyltransferase

acts preferentially on those CG sequences that arebase-paired with a CG sequence that is already

methylated

Formation of 5-methyl-cytosine occurs by methylation of




Formation of 5 methyl cytosine occurs by methylation of

a cytosine base in the DNA double helix

How DNA methylation patterns are faithfully




How DNA methylation patterns are faithfully

inherited

• Because of the existence of a methyl-directed methylating

enzyme (the maintenance methyltransferase), once a pattern of

DNA methylation is established, that pattern of methylation is

inherited in the progeny DNA



DNA methylation has several uses in the

vertebrate cell (?)

G i



• This can be faithfully passed on to progeny cells

However, two general mechanisms have emerged.

1. DNA methylation of the promoter region of a gene or of its

regulatory sequences can interfere directly with

the binding of proteins required for transcription initiation.2. The cell has a repertoire of proteins that specifically bind to

methylated DNA thereby blocking access of other proteins.

• Detail mechanism of gene repression via DNA

methylation is still under investigation

Gene repression

Multiple mechanisms contribute to stable gene




repression

Histone reader and

writer proteins,

under the direction

of gene regulatory

proteins, establish a

repressive form of

chromatin



Genomic Imprinting

Phenomenon in which a gene is either expressed

or not expressed in the offspring depending on

which parent it is inherited from



e.g. gene for insulin-like growth factor-2 (Igf2)

• Igf2 is required for prenatal growth, and mice that do

not express lgf2 at all are born half the size of normal

mice.

• However, only the paternal copy of lgf2 is transcribed,and only this gene copy matters for the phenotype.

• This is an example of maternal imprinting: copy of the

gene derived from mother is inactive

• As a result, mice with a mutated paternally derived

lgf2 gene are stunted, while mice with a mutated

maternally derived Igf2 gene are normal



• Imprinted genes are expressed as if there were only

one copy of the gene present in the cell even though

there are two.

• No changes are observed in the DNA sequences of

imprinted genes; that is inactive gene can be active or

inactive in the progeny, depending on whether it was

inherited from mom or dad.



If the DNA sequence of the gene does

not correlate with activity, what does ?

During the development of gametes, methyl groups

are added to the DNA in the regulatory regions of imprinted genes in one sex only.

DNA of genes that are shut down for an entire

lifetime are usually highly methylated

Gender-specific silencing of genes



p g g

and whole chromosomes

• Genomic imprinting explains some unusual patterns

of inheritance

• Certain autosomal genes have unusual inheritance patterns

e.g. an igf2 allele is expressed in a mouse only if it is inheritedfrom the father:

maternal imprinting because the copy of the gene derived

from the mother is inactive.

• Conversely, a mouse H19 allele is expressed only if it is

inherited from the mother- paternal imprinting because the

paternal copy is inactive






X-chromosome Inactivation (XCI)

• Unlike the gene-poor Y chromosome, the Xchromosome contains over 1,000 genes that are essential for

proper development and cell viability.

• However, females carry two copies of the X chromosome,

resulting in a potentially toxic double dose of X-linked genes.

• To correct this imbalance, mammalian females have evolved a

unique mechanism of dosage compensation:

transcriptionally silence one of their two X chromosomes in a

complex and highly coordinated manner

Barr body (?):

The inactivated X chromosome then condenses into a

compact structure called a Barr body, and it is stably

maintained in a silent state



• Mutations that interfere with dosage compensation

are lethal: the correct ratio of X chromosome

to autosome (non-sex chromosome) gene products is

critical for survival.

Mammalian X-chromosome inactivation




• X-chromosome inactivation begins with the synthesis of XIST (X-inactivation

specific transcript) RNA from the XIC (X-inactivation center) locus.

• The association of XIST RNA with one of a female's two X chromosomes is

correlated with the condensation of that chromosome





expression can be controlled

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