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Chapter 7: Control of Chapter 7: Control of Gene ExpressionGene Expression
Control of Gene ExpressionControl of Gene Expression
Different cell types differ dramatically in structure and function
►same genome
►Cell differentiation depends on gene expression
Control of Gene ExpressionControl of Gene Expression
Evidence for preservation of genome during cell differentiationEvidence for preservation of genome during cell differentiation
Control of Gene ExpressionControl of Gene Expression
Different Cell Types Synthesize Different Sets of Proteins►How many differences are there btwn any one cell type and another
►Many processes are common to all cells
►Some processes are cell specific
►Cell expresses ~10,000-20,000 of its 30,000 genes; level of expression of almost every gene varies from cell to cell
Control of gene ExpressionControl of gene Expression
Cells Can Change Expression of its Genes in Response to Cells Can Change Expression of its Genes in Response to External SignalsExternal Signals
► Different cell types respond in different ways to same extracellular signal= Different cell types respond in different ways to same extracellular signal= general feature of cell specializationgeneral feature of cell specialization
► Example: Liver and adipocyte cells respond differently to glucocorticoidExample: Liver and adipocyte cells respond differently to glucocorticoid
Liver Cell
Tyrosine aminotransferase
Adipocyte
Tyrosine aminotransferase
Control of Gene ExpressionControl of Gene Expression
For most genes transcription control is most important
Control of Gene ExpressionControl of Gene Expression
2 Fundamental Components to Transcriptional Gene 2 Fundamental Components to Transcriptional Gene RegulationRegulation1.1. Gene Regulatory ProteinsGene Regulatory Proteins
2.2. Short Stretches of DNA of Defined SequenceShort Stretches of DNA of Defined Sequence
Control of Gene ExpressionControl of Gene Expression
Outside of DNA Helix Read by Proteins
►GRP recognizes specific nucleotide sequence
►Information in form of:
H-bond acceptors
H-bond donors
Hydrophobic patches
►Bind to Major Groove
Control of Gene ExpressionControl of Gene Expression
GRPs bind to major groove where patterns for ea of fourGRPs bind to major groove where patterns for ea of four
base-pair arrangements are distinctbase-pair arrangements are distinct
Control of Gene ExpressionControl of Gene Expression
Geometry of Double Helix Depends on Nucleotide Sequence
Some nucleotide sequences cause DNA to bend
►AAAANNN
►If repeated every 10 bp DNA appears unusually curved
Control of Gene ExpressionControl of Gene Expression
DNA must be flexible for binding of GRPs
Control of Gene ExpresionControl of Gene Expresion
Short DNA Sequences Fundamental Components Short DNA Sequences Fundamental Components of of
Genetic SwitchesGenetic Switches1.1. GRP recognition sequence generally GRP recognition sequence generally << 20 bp 20 bp
2.2. Thousands of such DNA sequences identified ea of Thousands of such DNA sequences identified ea of which is recognized by different GRPwhich is recognized by different GRP
Control of Gene ExpressionControl of Gene Expression
GRP – DNA InteractionsGRP – DNA Interactions► Exact fit btwn DNA and proteinExact fit btwn DNA and protein► H-bonds, ionic bonds, H-bonds, ionic bonds,
hydrophobichydrophobic► >> 20 contacts 20 contacts► Tight and specificTight and specific
Control of Gene ExpressionControl of Gene Expression
Major Structural Motifs of GRPsMajor Structural Motifs of GRPs1.1. Helix-turn-helixHelix-turn-helix
2.2. HomeodomainHomeodomain
3.3. Zinc FingerZinc Finger
4.4. Leucine ZipperLeucine Zipper
5.5. Helix-Loop-HelixHelix-Loop-Helix
Control of Gene ExpressionControl of Gene Expression
Helix-Turn-HelixHelix-Turn-Helix► Most commonMost common► C-terminal helix= recognition C-terminal helix= recognition
helixhelix► aa in recognition helix define aa in recognition helix define
specificityspecificity► Structure of GRP varies outside Structure of GRP varies outside
HTH; HTH presented in unique HTH; HTH presented in unique wayway
Control of Gene ExpressionControl of Gene Expression
HomeodomainHomeodomain► Special type of helix-turn-helixSpecial type of helix-turn-helix► Conserved stretch of 60 aaConserved stretch of 60 aa► HTH motif always surrounded by same structure- homeodomainHTH motif always surrounded by same structure- homeodomain► Master regulators of developmentMaster regulators of development
Control of Gene ExpressionControl of Gene Expression
Zinc Finger ProteinsZinc Finger Proteins1.1. αα helix and helix and ββ sheet sheet
2.2. (2) (2) αα helices helices
Control of Gene ExpressionControl of Gene Expression
Leucine ZipperLeucine Zipper► ClothespinClothespin► Helices held together by Helices held together by
shortshort
coiled coil region of coiled coil region of hydrophobichydrophobic
residues often leucinesresidues often leucines
Control of Gene ExpressionControl of Gene ExpressionHelix-Loop-HelixHelix-Loop-Helix► Short Short αα helix connected to another via loop helix connected to another via loop► Flexible loop for packingFlexible loop for packing
Control of Gene ExpressionControl of Gene Expression
HeterodimerizationHeterodimerization► Enhances the repertoire of DNA binding specificitiesEnhances the repertoire of DNA binding specificities► Combinatorial controlCombinatorial control
Control of Gene ExpressionControl of Gene Expression
Is it possible to predict DNA sequence to Is it possible to predict DNA sequence to which GRP’s bind?which GRP’s bind?
Control of Gene ExpressionControl of Gene Expression
Gel Mobility Shift Assay to Detect GRPsGel Mobility Shift Assay to Detect GRPs► effect of a bound protein on the migration of DNA in an electric fieldeffect of a bound protein on the migration of DNA in an electric field
Control of Gene ExpressionControl of Gene Expression
DNA Affinity Chromatography to Purify GRPsDNA Affinity Chromatography to Purify GRPs
Purification of GRP Purification of GRP >> 10,000X 10,000X
Control of Gene ExpressionControl of Gene Expression
How do we determine the sequence to which a particular GRP binds?
Control of Gene ExpressionControl of Gene Expression
Chromatin Immunoprecipitation
►Identifies sequences occupied by GRPs
in living cells
►Used to identify direct targets of GRPs
How Genetic Switches WorkHow Genetic Switches Work
Tryptophan OperonTryptophan Operon
Operon= a cluster of genes transcribed as a single mRNA
Operator = short region of DNA in bact. that controls transcription of an adjacent gene
How Genetic Switches WorkHow Genetic Switches Work
Tryptophan Repressor = a Simple On/Off SwitchTryptophan Repressor = a Simple On/Off Switch
How Genetic Switches WorkHow Genetic Switches Work
Repressor= protein binds to DNA to prevent transcription of adjacent gene
Activator = protein that binds to DNA and promotes the transcription of adjacent gene
How Genetic Switches WorkHow Genetic Switches Work
CAP= Catabolite Activator Protein
►Promotes transcription of genes that enable E. coli to use
alternative carbon sources when glucose is not available
► glucose cAMP
►cAMP binds to CAP enabling CAP to bind to sequences near
target promoters to promote transcription
How Genetic Switches WorkHow Genetic Switches Work
Lac Operon- under the control of transcriptional activator and transcriptional repressor
More complicated genetic switches combine positive and negative controls
How Genetic Switches WorkHow Genetic Switches Work
Regulation of Transcription in Eukaryotes is More ComplexRegulation of Transcription in Eukaryotes is More Complex 1.1. GRPs can act even when positioned 1000’s bp away from GRPs can act even when positioned 1000’s bp away from
promoterpromoter
2.2. RNA Pol II cannot initiate transcription on its own, requires GTFsRNA Pol II cannot initiate transcription on its own, requires GTFs
3.3. Packaging of DNA in chromainPackaging of DNA in chromain
How Genetic Switches WorkHow Genetic Switches Work
Eucaryotic Gene Control Region
►Promoter and all regulatory sequences to which GRPs bind to control transcription
►> 50,000 bp, not unusual
►Packaged in nucleosomes and higher order forms of chromatin
How Genetic Switches WorkHow Genetic Switches Work
Eucaryotic GRPs
►5-10% of human genome
►Vary from one control region to next
►Present in sm amts, <0.01% total protein
►Most recognize specific DNA sequences; others assemble on other DNA bound proteins
►Allow genes to be turned on and off very specifically
How Genetic Switches WorkHow Genetic Switches WorkEucaryotic Gene Activator Proteins Promote Eucaryotic Gene Activator Proteins Promote
Assembly Assembly
of RNA Polymerase and GTFs at Transcription of RNA Polymerase and GTFs at Transcription StartStart
Gene Activator Proteins have Modular Design:
► DNA Binding Domain
► Activator Domain
How Genetic Switches WorkHow Genetic Switches Work
Mechanism of Gene Activator Proteins Varied but All Promote Assembly of GTFs and RNA Pol
►Interact w/ initiation complex to recruit RNA Pol
►Interact directly w/RNA Pol and GTFs
►Change chromatin structure around promoter
How Genetic Switches WorkHow Genetic Switches Work
GRPs can affect: GRPs can affect: ► prescribed ordered assembly of GTFs and RNA Polymeraseprescribed ordered assembly of GTFs and RNA Polymerase► Recruitment of RNA Polymerase holoenzyme to promoterRecruitment of RNA Polymerase holoenzyme to promoter
How Genetic Switches WorkHow Genetic Switches Work
Gene Activator Proteins Promote Assembly of GTFs and RNA Pol By
►Modification of Local Chromatin Structure Recruiting
histone acetyl transferases
histone remodeling complexes
How Genetic Switches WorkHow Genetic Switches Work
Gene Activator Proteins Work Synergistically
How Genetic Switches WorkHow Genetic Switches Work
EX: Complexity of How Gene Activator Proteins May Ultimately Increase Transcription Rate
How Genetic Switches WorkHow Genetic Switches Work
Eucaryotic Repressors Inhibit Transcription in Variety of Ways
How Genetic Switches WorkHow Genetic Switches Work
Eucaryotic GRPs and Combinatorial Control
►Function as unit to generate complexes whose
function depends on final assembly of all
components
►Not designated activators or repressors
►DNA acts as nucleation site for assembly
►Can participate in > one type of reg. complex
►Coactivators and corepressors
►enhancesome
How Genetic Switches WorkHow Genetic Switches Work
Eve-skipped gene is a complex multicomponent genetic switch in drosophilia
►Drosophilia development
►Eve expressed when embryo single giant multinucleated cell
►Cytoplasm=mixture of GRPs distributed unevenly along length of embryo
►Nuclei originally identical but later express diff genes cuz exposed to diff GRPs
How Genetic Switches WorkHow Genetic Switches Work
Eve Expression
►Regulatory sequence reads conc of GRPs at ea position along length of embryo
►Expressed in 7 stripes 5-6 nuclei wide precisely positioned along anterior- posterior axis
How Genetic Switches WorkHow Genetic Switches Work
Regulatory Region of Eve Gene
►~20,000 bp binds >20 proteins
►Series of regulatory modules
►Regulatory modules contain multiple reg sequences responsible for
specifying a particular stripe
How Genetic Switches WorkHow Genetic Switches Work
Expression of Stripe 2
►Dictated by 2 gene activator proteins and 2 gene repressor proteins
►Transcription occurs when activators Biocoid and Hunchback are high
and repressors Kruppel and Giant are low
How Genetic Switches WorkHow Genetic Switches Work
Combinatorial ControlCombinatorial Control► Heterodimerization of GRPs in solnHeterodimerization of GRPs in soln► Assembly of combos of GRPs into sm complexes on DNAAssembly of combos of GRPs into sm complexes on DNA► Many sets of grps bound simultaneous to effect transcriptionMany sets of grps bound simultaneous to effect transcription
How Genetic Switches WorkHow Genetic Switches Work
Simple regulatory modules= theme of Simple regulatory modules= theme of complex gene regulatory control complex gene regulatory control regions in mammalsregions in mammals
► 5-10% coding capacity of mam genome= 5-10% coding capacity of mam genome= GRPsGRPs
► Ea gene regulated by set of GRPsEa gene regulated by set of GRPs► Ea protein is product of gene that is in turn Ea protein is product of gene that is in turn
regulated by set of other proteinsregulated by set of other proteins► Activity of GRPs regulatedActivity of GRPs regulated
How Genetic Switches WorkHow Genetic Switches Work
Regulation of Activity of GRPsRegulation of Activity of GRPs
How Genetic Switches WorkHow Genetic Switches Work
Human β-globin Gene
►Complex regulation- 2 step process
►Expressed only in RBC at specific time during development
►Possesses own set of GRPs but also under control of LCR
►Cells where no globin gene expressed gene cluster tightly pkged
►Higher order pkging decondensed in RBS
How Genetic Switches WorkHow Genetic Switches Work
LCR= regulatory seq that govern accessibility and LCR= regulatory seq that govern accessibility and expression of distant genes or gene clustersexpression of distant genes or gene clusters
► ββ-thalassemia= deletion in -thalassemia= deletion in ββ-globin LCR causing gene to remain -globin LCR causing gene to remain transcriptionally silent– transcriptionally silent–
► Many LCRs present in human genomeMany LCRs present in human genome
How Genetic Switches WorkHow Genetic Switches Work
Insulators or Boundary Sequences►Bind Specialized Proteins
►Regulatory compartmentalization (Define domains of gene expression)
►Buffer genes from repressing effects of heterochromatin
►Block effect of enhancers (insulator must be btwn enhancer and promoter)
►Mechanism not understood
How Genetic Switches WorkHow Genetic Switches Work
Bacteria use interchangeable sigma subunits to help Bacteria use interchangeable sigma subunits to help regulate transcription while eucaryotes use (3) diff RNA regulate transcription while eucaryotes use (3) diff RNA PolPol
How Genetic Switches WorkHow Genetic Switches Work
Procaryotes vs Eucaryotes?Procaryotes vs Eucaryotes?
Molecular Genetic Mechanisms of Molecular Genetic Mechanisms of Specialized Cell TypesSpecialized Cell Types
Cell Memory= prerequisite for the creation of Cell Memory= prerequisite for the creation of organized tissues and the maintenance of stably organized tissues and the maintenance of stably differentiated cell typesdifferentiated cell types
Molecular Genetic Mechanisms of Molecular Genetic Mechanisms of Specialized Cell TypesSpecialized Cell Types
Gene Expression and Specialized Cell TypesGene Expression and Specialized Cell Types► Environmental effectsEnvironmental effects► Cell memory Cell memory ► Logic circuits Logic circuits
differentiatedifferentiate
keep timekeep time
remember events of the pastremember events of the past
adjust gene expression over whole chromosomeadjust gene expression over whole chromosome
Molecular Genetic Mechanisms of Molecular Genetic Mechanisms of Specialized Cell TypesSpecialized Cell Types
DNA rearrangements mediate phase variation in bacteriaDNA rearrangements mediate phase variation in bacteria
Site Specific Recombination at promoterSite Specific Recombination at promoter
Molecular Genetic Mechanisms of Molecular Genetic Mechanisms of Specialized Cell TypesSpecialized Cell Types
Rearrangements at the Mat locus determines Rearrangements at the Mat locus determines
mating type in budding yeastmating type in budding yeast
Molecular Genetic Mechanisms of Molecular Genetic Mechanisms of Specialized Cell TypesSpecialized Cell Types
Positive Feedback Loops Involving GRPs can Create Cell Memory
Lambda Repressor and Cro GRPs Maintain Mode of Growth of Lambda Phage
Molecular Mechanisms of Molecular Mechanisms of Specialized Cell TypesSpecialized Cell Types
Heritable State of Bacteriophage LambdaHeritable State of Bacteriophage Lambda► Switch controls flip-flop btwn lytic and lysogenic Switch controls flip-flop btwn lytic and lysogenic
statestate► Governed by two proteins that repress ea other’s Governed by two proteins that repress ea other’s
synthesis synthesis Lambda repressor protein cILambda repressor protein cI CroCro
► 50 genes in genome50 genes in genome
Molecular Mechanisms of Molecular Mechanisms of Specialized Cell TypesSpecialized Cell Types
Lysogenic- bacteriophage DNA integrated into host genome Lysogenic- bacteriophage DNA integrated into host genome
Lytic- virus multiplies, capsid protein translated and encapsulates virus Lytic- virus multiplies, capsid protein translated and encapsulates virus which exits host cell and in so doing lysis cellwhich exits host cell and in so doing lysis cell
Molecular Mechanisms of Molecular Mechanisms of Specialized Cell TypesSpecialized Cell Types
Prophage or lysogenic state= lambda repressor occupies operator synthesis of Cro and its own synthesis
Lytic State= Cro occupies diff site on operator synthesis of cI and synthesis its own synthesis to multiply and exit host
Molecular Mechanisms of Molecular Mechanisms of Specialized Cell TypesSpecialized Cell Types
Internal rhythmsInternal rhythms► Governs behavior at diff times of Governs behavior at diff times of
dayday► Established by day/night cycleEstablished by day/night cycle► Operates via transcriptional Operates via transcriptional
feedback loopfeedback loop► Resetting clock= destruction of a Resetting clock= destruction of a
key GRPkey GRP
Molecular Mechanisms of Molecular Mechanisms of Specialized Cell TypesSpecialized Cell Types
Combinatorial control
►Expression of set of genes can be coordinated by single protein
►Effect of single GRP can be decisive
Molecular Mechanisms of Molecular Mechanisms of Specialized Cell TypesSpecialized Cell Types
►Expression of critical GRP can trigger expression of entire battery of downstream genes
►Ability to switch many genes on or off coordinately impt to cell differentiation
►Conversion of one cell type to another by single GRP emphasizes how dramatic differences in cell types in size, shape, chemistry and function can be produced by differences in gene expression
Molecular Mechanisms of Molecular Mechanisms of Specialized Cell TypesSpecialized Cell Types
Combinatorial Gene Control Creates
Many Different Cell Types in Eucaryotes
Molecular Mechanisms of Molecular Mechanisms of Specialized Cell TypesSpecialized Cell Types
Combinatorial Gene Control Creates
Many Different Cell Types in Eucaryotes
Molecular Mechanisms of Molecular Mechanisms of Specialized Cell TypesSpecialized Cell Types
Formation of Entire Organ Coordinated by Single GRP
►Ey coordinates development of Drosophilia eye
Molecular Mechanism ofMolecular Mechanism ofSpecialized Cell TypesSpecialized Cell Types
Transmitting Stable Patterns of Gene Expression Transmitting Stable Patterns of Gene Expression ► Positive feedback loops; GRP activates own expressionPositive feedback loops; GRP activates own expression► Inhibiting expression an inhibitor to activate and maintain Inhibiting expression an inhibitor to activate and maintain
own expressionown expression► Propagation of chromatin structurePropagation of chromatin structure
Molecular Mechanism ofMolecular Mechanism ofSpecialized Cell TypesSpecialized Cell Types
Chromatin states
►heritable
►establish and preserve patterns of gene expression
Molecular Mechanism ofMolecular Mechanism ofSpecialized Cell TypesSpecialized Cell Types
Mechanisms of Dosage CompensationMechanisms of Dosage Compensation► X-inactivation- humansX-inactivation- humans► Male specific “up-regulation” of transcription- Male specific “up-regulation” of transcription-
DrosophiliaDrosophilia► Two-fold “down regulation” of X chromosome Two-fold “down regulation” of X chromosome
transcription- wormtranscription- worm
Molecular Mechanism ofMolecular Mechanism ofSpecialized Cell TypesSpecialized Cell Types
X-inactivation Center
106 nucleotide pairs
Lg regulatory center
Seeds formation of heterochromatin and facilitates its spread
XIST RNA coats inactive chromosome
Molecular Mechanism ofMolecular Mechanism ofSpecialized Cell TypesSpecialized Cell Types
Role of DNA Methylation in Gene ExpressionRole of DNA Methylation in Gene Expression► Patterns can be inheritedPatterns can be inherited► Reinforces transcriptional repression established by other Reinforces transcriptional repression established by other
mechanismsmechanisms► Lock genes in silent state- preventing leaky transcription (10Lock genes in silent state- preventing leaky transcription (106 6 ))► Maintains integrity of genomeMaintains integrity of genome► Genomic imprintingGenomic imprinting
Molecular Mechanism ofMolecular Mechanism ofSpecialized Cell TypesSpecialized Cell Types
Genomic Imprinting
When the expression of a gene is dependent upon whether it is maternally or paternally inherited
Molecular Mechanism ofMolecular Mechanism ofSpecialized Cell TypesSpecialized Cell Types
Maternal: CTCF binds to insulator preventing enhancer from interacting w/ Igf2 gene= no expression
Paternal: methylation at insulator site prevents CTCF binding allowing enhancer to interact w/ Igf2 gene = transcription
Molecular Mechanism ofMolecular Mechanism ofSpecialized Cell TypesSpecialized Cell Types
CG Islands
►Deamination of methylated C’s nonmutant T
►Deamination of methylated C’s U which is repaired
►Over evolutionary time 3 out of 4 CGs lost in this way
►Remaining CG unevenly distributed
Posttranscriptional RegulationPosttranscriptional Regulation
Posttranscriptional Controls
►Operate after RNA Pol initiated transcription
►Less common than transcriptional control but
essential in many cases
Posttranscriptional RegulationPosttranscriptional Regulation
Transcriptional AttenuationTranscriptional Attenuation► Premature termination of transcriptionPremature termination of transcription► mRNA structure interacts w/ RNA Pol in manner that aborts transcriptionmRNA structure interacts w/ RNA Pol in manner that aborts transcription► Premature termination can be prevented by proteins that bind to mRNA stem Premature termination can be prevented by proteins that bind to mRNA stem
looploop
Posttranscriptional RegulationPosttranscriptional Regulation
Alternative Splicing
►Different ways to splice primary transcript resulting in different polypeptides
►Protein complexity can exceed number of genes
►Regulation both positive and negative
Posttranscriptional RegulationPosttranscriptional Regulation
Regulation of RNA cleavage site and Poly-A-additionRegulation of RNA cleavage site and Poly-A-addition► Changes COOH terminus Changes COOH terminus ► Ex: membrane bound or secreted antibody molecules by B lymphocytesEx: membrane bound or secreted antibody molecules by B lymphocytes
Posttranscriptional RegulationPosttranscriptional Regulation
RNA EditingRNA Editing► Posttranscriptional alternation in mRNA sequencePosttranscriptional alternation in mRNA sequence► Tranpanosome mitochondrial sequences insertionTranpanosome mitochondrial sequences insertion
of U’sof U’s► Plant mitochondrial genes C’s changed to U’sPlant mitochondrial genes C’s changed to U’s► Mediated by guide RNAs w/ 5’ end comple-Mediated by guide RNAs w/ 5’ end comple-
mentary to transcriptmentary to transcript► Mammals deamination of adenine to inosineMammals deamination of adenine to inosine
which pairs w/ C; mediated by ADARs thatwhich pairs w/ C; mediated by ADARs that
recognize ds RNA structurerecognize ds RNA structure
Posttranscriptional RegulationPosttranscriptional Regulation
Regulation of nuclear exportRegulation of nuclear export