Chapter9- TranscriptionalControlofGeneExpression

Chapter9- TranscriptionalControlofGeneExpression

9.1ControlofGeneExpressioninBacteria9.2OverviewofEukaryoticGeneControl9.3RNAPolymeraseIIPromotersandGeneralTranscriptionFactors9.4RegulatorySequencesinProtein-CodingGenesandtheProteinsThroughWhichTheyFunction9.5MolecularMechanismsofTranscriptionRepressionandActivation9.6RegulationofTranscription-FactorActivity9.7EpigeneticRegulationofTranscription9.8OtherEukaryoticTranscriptionSystems

InteractwithoutsideworldBehavior– Output– Change

Environmentalchange– Behavioralchange…

CelllevelMoleculelevel

Organlevel

Behaviorlevel

Interactwithoutsideworld

CelllevelMoleculelevel

What’sthebasicproductofcell?

ProteinsGene

Genome

Howtoregulatetheproteinexpression?

Environmentalchanges

1. Transcription

1. Translation

1. Proteindegradation

1. RNAdegradation

Interactwithoutsideworld

CelllevelMoleculelevel

What’sthebasicproductofcell?

ProteinsGene

Genome

What’sthemostefficientwaytoregulatetheproteinexpression?

Environmentalchanges

Interactwithoutsideworld

CelllevelMoleculelevel

What’sthebasicproductofcell?

ProteinsGene

Genome

What’sthemostefficientwaytoregulatetheproteinexpression?

EnvironmentalchangesTranscriptionalregulation!Howpercentageisit?

Contributionsofthemajorprocessesthatregulateproteinconcentrations.

Whenisthetranscriptionalregulationimportant?

• Interactwithoutsideworld• ?• Formakingyourownshape…

Developmentalprocess…

DNAmicroarray

Geneexpressionchangesinmicroarraystudy

Phenotypesofmutationsingenesencodingtranscriptionfactors.

• Improperregulationofmechanismscontrollingtranscriptioncausespathologicalprocesses.

• (a)AhumanHOXD13 genedominantmutation:• polydactyly– developmentofextradigits

• (b)DrosophilaUbx genehomozygousrecessivemutations:

• preventUbx expressioninthethirdthoracicsegment

• transformssegment,whichnormallyhasabalancingorgancalledahaltere,intoasecondcopyofthethoracicsegmentthatdevelopswings

• (c)InactivatingmutationsinbothcopiesofthreeArabidopsisthalianafloralorgan–identitygenes:

• transformthenormalflowerpartsintoleaflike structures

• Mutationsaffectmasterregulatorytranscriptionfactorsthatregulatemultiplegenes,includingmanygenesencodingothertranscriptionfactors.

Howtoregulatethetranscription?

TranscriptionalregulationinBacteria

Learningtheprinciples…

TranscriptionalControlofGeneExpression

9.1ControlofGeneExpressioninBacteria• Prokaryotegeneexpression– regulatedprimarilybymechanismsthatcontrolgenetranscription

• Thelacoperonandsomeotherbacterialgenesareregulatedbyactivatorproteinsthatbindnexttoapromoter andactivateRNApolymerase.

• TranscriptioninitiationbyE.coliRNApolymerasescanberegulatedbyrepressorsandactivatorsthatbindnearorupto100basesupstreamofthetranscriptionstartsite.

• Transcriptioninbacteriacanberegulatedbycontroloftranscriptionalelongationinthepromoter-proximalregion.

Tounderstandthetranscriptionalregulationofanorganism,

• Weshouldknowtheorganism.

• Weshouldknowtheenvironmentaroundtheorganism.

Interaction!

E.coli

• Escherichiacoli• Escherichiacoli isa gram-negative, facultativelyanaerobic, rod-shaped, coliformbacterium ofthe genus Escherichia thatiscommonlyfoundinthelower intestine of warm-blooded organisms(endotherms)

Symbiosis

Gutflora (gutmicrobiota,or gastrointestinalmicrobiota)isthecomplexcommunityof microorganisms thatliveinthe digestivetracts ofhumansandotheranimals,includinginsects.

Intestinal bacteria alsoplayaroleinsynthesizing vitaminB and vitaminK aswellasmetabolizing bileacids, sterols,and xenobiotics.

JustlearnabouttheE.coli

• Gram-negative

• Facultatively anaerobic

Gram-negativebacteria areagroupof bacteria thatdonotretainthe crystalviolet stain usedinthe Gramstaining methodofbacterialdifferentiation.

JustlearnabouttheE.coli

• Gram-negative

• Facultatively anaerobic

Gram-negativebacteria areagroupof bacteria thatdonotretainthe crystalviolet stain usedinthe Gramstaining methodofbacterialdifferentiation.

JustlearnabouttheE.coli

• Gram-negative

• Facultatively anaerobic

Gram-negativebacteria areagroupof bacteria thatdonotretainthe crystalviolet stain usedinthe Gramstaining methodofbacterialdifferentiation.

A facultativeanaerobe isan organism thatmakes ATP by aerobicrespiration if oxygen ispresent,butiscapableofswitchingto fermentation or anaerobicrespiration ifoxygenisabsent.

RegulationoftranscriptionfromthelacoperonofE.coli.

• E.coliinanenvironmentthatlackslactose:

• E.coliinanenvironmentcontainingbothlactoseandglucose:

• E.coliinanenvironmentcontaininglactosebutlackingglucose:

Lactosemetabolism

RegulationoftranscriptionfromthelacoperonofE.coli.

• E.coliinanenvironmentthatlackslactose:represslacmRNAsynthesissothatcellularenergyisnotwastedsynthesizingunnecessaryenzymes.

• E.coliinanenvironmentcontainingbothlactoseandglucose:cellspreferentiallymetabolizeglucose,thecentralmoleculeofcarbohydratemetabolism.

• E.coliinanenvironmentcontaininglactosebutlackingglucose:cellsmetabolizelactoseatahighrateonlywhenlactoseispresentandglucoseislargelydepletedfromthemedium.

Transcriptionalregulation

• Thinkabouttranscription

HowtoblockthebindingofRNApolymerase?

Howtocontrolthetranscription?1.Blockthetranscription

Howtocontrolthetranscription?1. Blockthetranscription2. Activatethetranscription

Transcriptionalregulation

• Cis-actingelement• Trans-actingelement

Promoter

Protein

RegulationoftranscriptionfromthelacoperonofE.coli.

• CAPsite– bindscataboliteactivatorprotein.• lacpromoter– bindstheσ70-RNApolymerasecomplex

• lacoperator– bindslacrepressor

cAMP-bound cataboliteactivatorprotein(CAP)

RegulationoftranscriptionfromthelacoperonofE.coli.

• Tetramericlacrepressor:bindstotheprimarylacoperator(O1)andoneoftwosecondaryoperators(O2orO3)simultaneously.Thetwostructuresareinequilibrium.

Lacoperon

https://www.youtube.com/watch?v=oBwtxdI1zvk

• MostE.colipromotersinteractwithσ70-RNApolymerase,themajorformofthebacterialenzyme.

DNAloopingpermitsinteractionofboundNtrC andσ54-RNApolymerase.

regulatedsolelybyactivatorsthatbindtoenhancersitesgenerallylocated80–160bpupstreamfromthetranscriptionstartsite

• NtrC σ54-activator:• NtrC protein(nitrogenregulatory

proteinC)– stimulatestranscriptionoftheglnA gene

• glnA gene– encodesglutaminesynthetase,whichsynthesizesglutamine,thecentralmoleculeofnitrogenmetabolism,fromglutamicacidandammonia

• NtrC ATPhydrolysis– requiredforactivationofσ54-RNApolymerase

Enhancer

The σ54-RNA polymerase binds to the glnA promoter but does not melt the DNA strands and initiate transcription until it is activated by NtrC, a dimeric protein.

Howtodetectamountofasubstanceandregulatetheamount?

?

Regulator SubA synthesisenzyme

Howtodetectasubstanceandregulate?

BindingtoSubA SubA synthesisenzyme

Howtodetectasubstanceandregulate?

BindingtoSubA SubA synthesisenzyme

Howtodetectasubstanceandregulate?

BindingtoSubA SubA productiongeneactivator

Production

Activate

Howtodetectasubstanceandregulate?

BindingtoSubA

Production

Activate

Two-componentregulatorysystems.

• Manybacterialresponsesarecontrolledbytwo-componentregulatorysystems.

• (a)Lowcytoplasmicglutamineconcentration:• glutaminedissociatesfromNtrB.• NtrB conformationalchangeactivatesaproteinkinasetransmitterdomainthattransfersanATPγ-phosphatetoaconservedhistidine(H)inthedomain.

• Phosphate– thentransferredtoanasparticacid(D)intheregulatorydomain oftheresponseregulatorNtrC

• PhosphorylationconvertsNtrC intoitsactivatedform,whichbindstheenhancersitesupstreamoftheglnApromoter.

Generaltwo-componentsignalingsystem

• (b)Two-componenthistidyl-aspartylphospho-relayregulatorysystemsinbacteriaandplants– generalorganization

Otherwaytoregulatethetranscription

Transcriptionalregulation

• Transcriptioninitiation,then?

Elongation&Termination

Trp operon

But,stillleakofexpression

Calledas“attenuation”

Howtoblocktheexpressioncompletely?

TranscriptionalcontrolbyregulationofRNApolymeraseelongationandterminationintheE.colitrp operon.

• Ribosometranslatesquicklythroughregion1intoregion2,blockingregion2basepairingwithregion3.

• Region3basepairswithregion4.• 3–4stem-loopandfollowingseriesofuracils – signal

forterminationofRNApolymerasetranscriptionofrestofoperon

• Region3issequesteredinthe2–3stem-loopandcannotbase-pairwithregion4.

• Transcriptionofthetrp operoncontinues.

1.Nocodingforenzymes(~140nts)

2.Trp-Trp codons

3.

4.Indicatetermination

Riboswitchcontroloftranscriptiontermination inB.subtilis. Bacillussubtilis,knownalsoasthe haybacillus or grassbacillus,is

a Gram-positive, catalase-positive bacterium

xpt-pbuX operon(encodesenzymesinvolvedinpurinesynthesis)

TranscriptionalControlofGeneExpression

9.2OverviewofEukaryoticGeneControl• Transcriptionalcontrolistheprimarymeansofregulatinggeneexpression

ineukaryotes.• Eukaryotescontainthreetypes ofnuclearRNApolymerases:

– RNApolymeraseIsynthesizesonlypre-rRNA.– RNApolymeraseIIsynthesizesmRNAs,someofthesmallnuclear

RNAsthatparticipateinmRNAsplicing,andmicro- andsmallinterferingRNAs(miRNAs andsiRNAs)thatregulatethetranslationandstabilityofmRNAs.

– RNApolymeraseIIIsynthesizestRNAs,5SrRNA,andseveralothersmallstableRNAs.

Fundamentaldifferencebetweeneukaryoticandprokaryoticchromatin?

• SinglehumancellDNAmeasuresabout2mintotallength,andiscontainedwithinnucleiwithdiametersoflessthan10μm – acompactionratioofgreaterthan105 to1.

• EachchromosomeconsistsofasingleDNAmolecule(aslongas280Mbinhumans),organizedintoincreasinglevelsofcondensationfromnucleosomestohigherorderchromatinfoldingbyhistoneandnonhistone proteins.

• AnygivenportionofhighlycompactedDNAcanbeaccessedfortranscription,replication,andrepairofdamagewithoutthelongDNAmoleculesbecomingtangledorbroken.

• HighereukaryoticDNAconsistsofuniqueandrepeatedsequences:

• Onlyabout1.5percentofhumanDNAencodesproteinsandfunctionalRNAs.

• Theremainderincludesregulatorysequencesthatcontrolgeneexpressionandintrons.

• About45percentofhumanDNAisderivedfrommobileDNAelements,geneticsymbiontsthathavecontributedtotheevolutionofcontemporarygenomes.

Overviewofeukaryotictranscriptionalcontrol.

• Chromatincondensationinactivatesgenetranscription:• BlocksRNApolymerasesandgeneraltranscriptionfactors

frominteractingwithgenepromoters• Repressorproteins:

• Maybindtotranscription-controlelementstoinhibittranscriptioninitiationbyPolII

• Mayinteractwithmultiproteinco-repressorcomplexestocondensechromatin

• Pioneertranscriptionfactor:• Bindstoaspecificregulatorysequencewithinthe

condensedchromatin• Interactswithchromatin-remodelingenzymesandhistone

acetylases thatdecondense thechromatin,makingitaccessibletoRNApolymeraseIIandgeneraltranscriptionfactors.

• Activatorproteins:• Bindtospecifictranscription-controlelementsinboth

promoter-proximalsitesanddistantenhancers• Interactwithoneanotherandwiththemultisubunit

MediatorcomplextoassemblegeneraltranscriptionfactorsandRNApolymeraseII(PolII)onpromoters

• Transcriptionalactivation:• PolIIinitiatestranscription.• PolIIpausesaftertranscribingfewerthan100nucleotides

becauseofactionoftheelongationinhibitorNELF(negativeelongationfactor)associatedwithDSIF(DRBsensitivity-inducingfactor).

• Activators:• PromoteassociationofthePolII-NELF-DSIFcomplexwith

elongationfactorP-TEFb (cyclinT-CDK9[kinase]),whichreleasesNELF

• NELFreleaseallowsresumptionofRNAtranscription.

Pax 6gene

Pax6protein– requiredfordevelopmentoftheeye,certainregionsofthebrainandspinalcord,andthepancreascellsthatsecretehormonessuchasinsulin

regulation at each step m gene expression have been found, control of transcript ion initiation and elongation-the first rwo steps-arc the most important mechanisms for determining whether most genes are expressed and how much of the en-coded mRNAs and, consequently, proteins are produced. The molecular mechanisms that regulate transcription initiation and elongation are critical to numerous biological phenomena, in-cluding the development of a multicellular organism from a single fertilized egg cell as mentioned above, the immune re-sponses that protect us from pathogenic microorganisms, and

(a)

(b)

Haltere

Normal

(c)

FIGURE 7·1 Phenotypes of mutations in genes encoding transcription factors. (a) A mutation that inactivates one copy of the Pax6 gene on either the maternal or paternal chromosome 9 results in failure to develop an iris, or aniridia. (b) Homozygous mutations that prevent expression of the Ubx gene in the third thoracic segment of Drosophila result in transformation of t he third segment, which normally has a balancing organ called a haltere, into a second copy of the thoracic segment that develops wings. (c) Mutations in Arabidopsis

280 CHAPTER 7 • Tran scriptional Control of Gene Expression

neurological processes such as learning and memory. When these regulatory mechanisms contro ll ing transcription function improperly, pathological processes may occur. For example, reduced activity of the Pax6 gene causes aniridia, failure to de-velop an iris (Figure 7-la) . Pax6 is a transcription factor that normally regulates transcription of genes involved in eye devel-opment. In other organisms, mutations in transcription factors cause an extra pair of wings ro develop in Drosophila (Figure 7-1 b), alter the structures of flowers in plants (Figure 7- l c), and are responsible for multiple other developmental abnormalities.

Ubxmutant

thaliana that inactivate both copies of t hree flora l organ-identity transform the normal parts of the f lower into leafl ike structures. In each case, these mutations affect master regulatory transcription factors that regulate multiple genes, including many genes encoding other transcription factors. [Part (a), left, Simon Fraser/Photo Researchers, Inc.; right, Visuals Unlimited. Part (b) from E. B. Lewis, 197B, Nature 276:565. Part (c) from D. Wiegel and E. M. Meyerowiu, 1994, Ce//78:203.]

A mutation that inactivates one copy of the Pax6 gene on either the maternal or paternal chromosome 9 results in failure to develop an urus, or aniridia.

Transcription-controlregionsofthemousePax6geneandtheorthologoushumanPAX6gene

• ThreealternativePax6promoters:• Transcription-controlregions(200–500bp long)regulateexpressionofPax6 atdifferentembryogenesistimesinindifferenttissues.

Betagalactosidase– asareporterfortranscriptionalregulation

Promoter Reporter

B-galactosidase

Experiment:tg mouseexpressingofabeta-galactosidasereportertransgenefusedtodifferentregionsofPas6gene

• Experiment:transgenicmouseexpressionofaβ-galactosidasereportertransgenefusedtodifferentregionsofPax6 gene(stainedwithX-galtorevealβ-galactosidase)

• (b)Embryo10.5daysafterfertilization– 8kbofmouseDNAupstreamfromexon0linkedtoreportertransgene

• Expression– inlenspit(LP)tissuethatwilldevelopintotheeyelensandintissuethatwilldevelopintothepancreas(P)

• (c)Embryo13.5daysafterfertilization– sequencebetweenexons4and5linkedtoβ-galactosidasereportergene

• Expression– Retinaandpancreas

(b)

(c)

ThehumanSALL1enhanceractivatesexpressionofareportergeneinlimbbudsofthedevelopingmouseembryo

HumanDNAsequence~500kbdownstreamoftheSALL1gene– highlyconservedbetweenhumans,mice,chickens,frog,andfish.

ThehumanSALL1enhanceractivatesexpressionofareportergeneinlimbbudsofthedevelopingmouseembryo

Beta-galpromoter

Plasmidvector

Luciferase

• Luciferase isagenerictermfortheclassofoxidativeenzymesthatproducebioluminescence,andisdistinctfromaphotoprotein.

ThreeRNApolymerases

HowtoseparateRNApolymerases

LiquidchromatographyseparatesandidentifiesthethreeeukaryoticRNApolymerases,eachwithitsownsensitivityto𝛂-amanitin.

AllthreeRNApolymerasesinaproteinextractfromculturedeukaryoticcellnucleiboundtoanegativelychargedDEAESephadex column

RNApolinhibitor

α-Amanitin (red)boundtoRNApolymeraseIIfrom Saccharomycescerevisiae (brewer'syeast).

α-Amanitin isanselectiveinhibitorof RNApolymeraseII and III. Thismechanismmakesitadeadlytoxin.

interfereswiththetranslocationofRNAandDNAneededtoemptythesiteforthenextroundofRNAsynthesis

LiquidchromatographyseparatesandidentifiesthethreeeukaryoticRNApolymerases,eachwithitsownsensitivityto𝛂-amanitin.

• RNApolymeraseI:• insensitive• locatedinthenucleolus• transcribesgenesencodingprecursorrRNA (pre-rRNA),whichisprocessedinto28S,5.8S,and18SrRNAs

• RNApolymeraseII:• verysensitive• transcribesallprotein-codinggenestoproducemRNAs

• RNApolymeraseIII:• intermediatesensitivity• transcribesgenesencodingtRNAs,5SrRNA,andanarrayofsmallstableRNAs,includingoneinvolvedinRNAsplicing(U6)andtheRNAcomponentofthesignalrecognitionparticle(SRP)

AllthreeRNApolymerasesinaproteinextractfromculturedeukaryoticcellnucleiboundtoanegativelychargedDEAESephadex column

Comparisonofthree-dimensionalstructuresofbacterialandeukaryoticRNApolymerases

• Fourmultisubunit RNApolymeraseshaveasimilaroveralldesign.• EachofthethreeeukaryoticRNApolymerasesismorecomplexthanE.coliRNApolymerase

• (a)BacterialRNApolymerase• (b)EukaryoticRNApolymeraseII(10ofthe12subunitsshowninmodel)• (c)YeastRNApolymeraseII– includingsubunits4and7thatextendfromthecoreportionoftheenzymeshownin(b)neartheregionoftheC-terminaldomainofthelargesubunit

Evolution?

RNApolIIsubunitsandstructure

https://www.youtube.com/watch?v=GdKfadJGId4

TheclampdomainofRPBI(RNApolB1=RNApolII).• DiffersfromfreemainlyinthepositionofaclampdomainintheRPB1subunit

• Clampdomainswingsoverthecleftbetweenthejawsofthepolymeraseduringformationofthetranscribingcomplex,trappingthetemplateDNAstrandandtranscript.

• Bindingoftheclampdomaintothe8–9-bpRNA-DNAhybrid– mayhelpcoupleclampclosuretothepresenceofRNA

• Mg2+ion– participatesincatalysisofphosphodiesterbondformation

• RPB2Walldomain– forcesthetemplateDNAenteringthejawsofthepolymerasetobendbeforeitexitsthepolymerase

• Bridgeαhelix:• extendsacrossthecleftinthepolymerase

• ispostulatedtobendandstraightenasthepolymerasetranslocates onebasedownthetemplatestrand

• Nontemplate strand– formsaflexiblesingle-strandedregionabovethecleft(notshown),extendingfromthreebasesdownstreamofthetemplatebase-pairedtothe3ʹbaseofthegrowingRNAtowherethetemplatestrandexitsthepolymerase

SchematicrepresentationofthesubunitstructureoftheE.coliRNAcorepolymeraseandyeastnuclearRNApolymerases.

Allthreeyeastpolymeraseshavefivecoresubunitshomologoustotheβ,βʹ,twoα,andω subunitsofE.coliRNApolymerase

RNApolymerasesIandIIIcontainthesametwononidentical α-likesubunits,whereasRNApolymeraseIIcontainstwoothernonidentical α-likesubunits.

• RNApolymeraseIIlargestsubunit(RPB1)– alsocontainsanessentialC-terminaldomain(CTD)

CTDofRNApolII

• RNAPolymeraseIIexistsintwoformsunphosphorylated andphosphorylated,IIAandIIOrespectively.

• ThemethodfortheelongationinitiationisdonebythephosphorylationofSerineatposition5(ser5),viaTFIIH.ThenewlyphosphorylatedSer5recruitsenzymestocapthe5'endofthenewlysynthesizedRNAandthe"3'processingfactorsto poly(A) sites".

CTDofRNApolIIinaction!Cracking the RNA polymerase II CTD codeSylvain Egloff, Shona Murphy <Find the paper!>

Antibodystainingdemonstratesthatthecarboxy-terminaldomainofRNApolymeraseIIisphosphorylated duringinvivotranscription.

• PhosphorylatedCTD(red)–stainedwithaphosphorylation-specificrabbitantibody

• Nonphosphorylated CTD(green)– stainedwithnonphosphorylation-specificgoatantibody

Whatcanyousee?

Polytene chromosomes areoversized chromosomes whichhavedevelopedfromstandardchromosomesandarecommonlyfoundinthesalivaryglandsof Drosophilamelanogaster.

Discussionwithfriends

• PleasefindthefunctionsofRNAs

• SummarizetherolesofCTDinRNAPolII-mediatedtranscription