Chapter 7: Control of Gene Expression. Control of Gene Expression Different cell types differ...

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


Evidence for preservation of genome during cell differentiationEvidence for preservation of genome during cell differentiation


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


For most genes transcription control is most important


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


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


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


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


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


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


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


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


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


Zinc Finger ProteinsZinc Finger Proteins1.1. αα helix and helix and ββ sheet sheet

2.2. (2) (2) αα helices helices


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


HeterodimerizationHeterodimerization► Enhances the repertoire of DNA binding specificitiesEnhances the repertoire of DNA binding specificities► Combinatorial controlCombinatorial control


Is it possible to predict DNA sequence to Is it possible to predict DNA sequence to which GRP’s bind?which GRP’s bind?


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


DNA Affinity Chromatography to Purify GRPsDNA Affinity Chromatography to Purify GRPs

Purification of GRP Purification of GRP >> 10,000X 10,000X


How do we determine the sequence to which a particular GRP binds?


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


Tryptophan Repressor = a Simple On/Off SwitchTryptophan Repressor = a Simple On/Off Switch


Repressor= protein binds to DNA to prevent transcription of adjacent gene

Activator = protein that binds to DNA and promotes the transcription of adjacent gene


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


Lac Operon- under the control of transcriptional activator and transcriptional repressor

More complicated genetic switches combine positive and negative controls


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


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


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


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


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


Gene Activator Proteins Promote Assembly of GTFs and RNA Pol By

►Modification of Local Chromatin Structure Recruiting

histone acetyl transferases

histone remodeling complexes


Gene Activator Proteins Work Synergistically


EX: Complexity of How Gene Activator Proteins May Ultimately Increase Transcription Rate


Eucaryotic Repressors Inhibit Transcription in Variety of Ways


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


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


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


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


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


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


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


Regulation of Activity of GRPsRegulation of Activity of GRPs


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


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


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


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


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


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


DNA rearrangements mediate phase variation in bacteriaDNA rearrangements mediate phase variation in bacteria

Site Specific Recombination at promoterSite Specific Recombination at promoter


Rearrangements at the Mat locus determines Rearrangements at the Mat locus determines

mating type in budding yeastmating type in budding yeast


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


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


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


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


Combinatorial control

►Expression of set of genes can be coordinated by single protein

►Effect of single GRP can be decisive


►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


Combinatorial Gene Control Creates

Many Different Cell Types in Eucaryotes


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


Chromatin states

►heritable

►establish and preserve patterns of gene expression


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


X-inactivation Center

106 nucleotide pairs

Lg regulatory center

Seeds formation of heterochromatin and facilitates its spread

XIST RNA coats inactive chromosome


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


Genomic Imprinting

When the expression of a gene is dependent upon whether it is maternally or paternally inherited


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


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


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


Alternative Splicing

►Different ways to splice primary transcript resulting in different polypeptides

►Protein complexity can exceed number of genes

►Regulation both positive and negative


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


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


Regulation of nuclear exportRegulation of nuclear export

Chapter 7: Control of Gene Expression. Control of Gene Expression Different cell types differ...

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