分子生物学Chapter 12 Eukaryotic Gene Regulation.docx
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分子生物学Chapter 12 Eukaryotic Gene Regulation.docx
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分子生物学Chapter12EukaryoticGeneRegulation
Chapter12EukaryoticGeneRegulation
Introduction
Thegenecontrolregionofatypicaleucaryoticgene.
vThepromoteristheDNAsequencewherethegeneraltranscriptionfactorsandthepolymeraseassemble.
Chapter12EukaryoticGeneRegulation
1.UpstreamElements(Promoter-proximalelement)
1.1Sitesforbasaltransription
HavealreadyseenindiscussionoftheRNApolIIBasalTranscriptionApparatusthatRNApolIIpromotersusuallycontain2elements:
vA.TheInrorInitiatorstartsiteofTranscription:
Py2CAPy5,withposn+1attheA
vB.TheTATAbox,~25bpupstreamof+1...forTBPbinding
1.2UpstreamElement(promoter-proximalelements)
vTheseshortsequencesarerecognizedbyProtein(trans-actingfactors)
vUpstreamFactors(activator)whichusuallyenhanceandactivatetranscriptionefficiencyand(sometimes)specificity
vWeusethetermpromoter-proximalelementsforcontrolregionslyingwithin100–200basepairsupstreamofthestartsite.
Otherexamplesareasfollows
vElementConSeqFactor
vTATAboxTATAAAATBP(TataBindingProtein)
vCAATboxGGCCAATCTCTF/NF1(CaaTbindingFactor/
NecrosisFactor1)
vGCboxGGGCGGSP1(StimulatoryProtein1)
vOctamerATTTGCATOct-1/Oct-2(Oct-2specificto
Lymphoidcells)
vATFGTGACGTATF
1.3ProteinFactors
vProteinFactorsrecognizetheUpstreamElementSites.
vTheUpstreamFactorsaremoreorlessubiquitousandhenceavailabletoanypromoterwitharecognitionsequenceinthepromoter.
(Sequenceelementpositionslargelyunimportant;canbeinterchangedbetweendifferentpromoters...distancebetweenelementsunimportant...==>loopingofDNAimportant;protein-proteininteractionsimportant)
UpstreamelementsmayberecognizedbymorethanoneFactor,egCAATbox:
v1).CTFfactorfamily,generatedbyalternativesplicingfromsinglegene
v2).CP1,CP2,CP3bindCAATboxesindifferentgenes...genespecificity
v3).C/EBPbindsGCAAT,ACFbindsCCAAT...sequencespecificity
Differentregulationbydifferentboundfactorsinsameupstreamelement.Example:
HistoneH2B
vInseaurchintestis,CAATisboundnormallyandH2Bisexpressed.
vInseaurchinembryos,CAATisboundbyCDP(CAATDisplacementProtein)whichpreventsbindingofnormalCAATbindingfactors->H2Bisnotexpressed
vOctamersequenceisanotherexampleofanUpstreamElementthatcanbeboundbymorethanoneUpstreamFactor:
v1).Oct-1factorisubiquitousandtheonlyOctfactorinnon-lymphoidcells
v2.)Oct-2factorinlymphoidcellsbindstoOctomertoactivateIGkappalightgene;thisgeneisnotactivatedbyOct-1factorinnon-lymphoidcells
(Thuspromotercontextisimportant...totalityofelementsandfactorsisimportant)
Chapter12EukaryoticGeneRegulation
2.Enhancers
vTranscriptionfrommanyeukaryoticpromoterscanbestimulated(activated)bycontrolelementscalledenhancers,whicharelocatedthousandsofbasepairsawayfromthestartsite.
vEnhancersareelementswhosepositionrelativetoInrisnotfixedandwhichfunctionineitherorientation.UsuallyenhancersactivateANYpromoterintheirvicinity.
vEnhancersinyeastarecalledUpstreamActivatorSequences(UASs)
Generalpatternofcis-actingcontrolelementsthatregulategeneexpressioninyeastandmulticellularorganisms
Forexample:
Thefirstenhancertobediscoveredthatstimulatedtranscriptionofeukaryoticgenes
v366-bpfragmentofthesimianvirus40(SV40)genome.
vFurtheranalysisofthisregionofSV40DNArevealedthata72-bpsequencelying≈100basepairsupstreamoftheSV40earlytranscription-startsitewasresponsibleforitsabilitytoenhancetranscription.
vEnhancerinvirusSV40whichiscomposedoftwocopiesof72bpsequence.
vTheSV40enhancercanstimulatestranscriptionfromallmammalianpromotersthathavebeentestedwhenitisinsertedineitherorientationanywhereonaplasmidcarryingthetestpromoter,evenwhenitisthousandsofbasepairsfromthestartsite.
vAnextensivelinkerscanningmutationalanalysisoftheSV40enhancerindicatedthatitiscomposedofmultipleindividualelementseachofwhichcontributestothetotalactivityoftheenhancer.Eachoftheseregulatoryelementsisaprotein-bindingsite.
Chapter12EukaryoticGeneRegulation
3.EukaryoticTranscriptionActivators
BiochemicalIsolationofTranscriptionFactors
InvitrotranscriptionactivationbySP1,whichbindsto10-bpGC-richsequences.
Invivoassayfortranscriptionfactoractivity
TranscriptionActivatorsAreModularProteinsComposedofDistinctFunctionalDomains
vTranscriptionfactorsmustperformatleastthefollowingtwofunctions:
1.bindtotargetEnhancerandUpstreamElementsequences
2.interactwithothermembersoftheTranscriptionApparatus
vThesecapabilitiesusuallyresideinseparatedomainsintheTranscriptionFactorprotein.
Example:
YeastActivatorGAL4
vThestructuralmodelofeukaryoticactivatorsisamodularoneinwhichoneormoreactivationdomainsisconnectedtoasequence-specificDNA-bindingdomainthroughrelativelyflexibleproteindomains.
DNA-BindingDomains
vEukaryotictranscriptionfactorscontainavarietyofstructuralmotifsthatinteractwithspecificDNAsequences.
vαhelicesintheDNA-bindingdomainofeukaryotictranscriptionfactorsareorientedsothattheylieinthemajorgrooveofDNAwhereproteinatomsmakespecifichydrogenbondsandvanderWaalsinteractionswithatomsintheDNA.Interactionswithsugar-phosphatebackboneatomsand,insomecases,withatomsintheDNAminorgroovealsocontributetobinding.
vAnumberofstructuralmotifspresentanαhelixtothemajorgroove.
vMostofthestructuralclassesofDNA-bindingdomainshavecharacteristicconsensusaminoacidsequences.
vThegenomesofhighereukaryotesmayencodedozensofclassesofDNA-bindingdomainsandliterallyhundredsoftranscriptionfactors.
HomeodomainProteins
Examples:
(1)TFIIIA,theRNAPolIIItranscriptionfactor,withC2H2zincfingerrepeated9times.
(2)SP1,with3copiesofC2H2zincfinger.
Usually,threeormoreC2H2zincfingersarerequiredforDNAbinding.
Theglucocorticoidreceptor,ahomodimericC4protein.Helicalregionsareshownasspirals,andβstrandsasbroadarrows.Twoαhelices(darkershade),oneineachmonomer,interactwiththeDNA.LikeallC4zinc-fingerhomodimers,thistranscriptionfactorhastwofoldrotationalsymmetry;thecenterofsymmetryisshownbytheyellowellipse.BlackcirclesindicateZn2+ions.
Leucinezippers
vLeucinezipperproteinscontainahydrophobicleucineresidueateveryseventhpositioninaregionthatisoftenattheC-terminalpartoftheDNA-bindingdomain
vTheseleucinesareresponsiblefordimerizationthroughinteractionbetweenthehydrophobicfacesoftheα-helices.Thisinteractionformsacoiled-coilstructure.
vInteractionwithasymmetricalDNArecognitionsitewiththezipperedproteinclamp
vTheNterminaldomainofeachhelixformasymmetricalstructureinwhicheachbasicdomainliesalongtheDNAinoppositedirectionsinteractingwithsymmetricalDNArecognitionsitesothatproteinineffectformaclamparoundDNA
Generegulatoryproteinsthatcontainaleucinezippermotifcanformeitherhomodimers,inwhichthetwomonomersareidentical,orheterodimers,inwhichthemonomersaredifferent
thehelix-loop-helixdomain
vInteractionofthehelix-loop-helixdomaininthehomodimericMaxproteinwithDNA.Thehelix-loophelixmotifextendsfromtheDNA-bindinghelicesontheleft(N-terminiofthemonomers)toapproximatelywherethechainsfirstcross;thisisfollowedimmediatelybyaleucine-zippercoiled-coilregioninthedimericprotein.
ActivationDomains
vActivationDomainsExhibitConsiderableStructuralDiversity.
vAnactivationdomainisapolypeptidesequencethatactivatestranscriptionwhenitisfusedtoaDNA-bindingdomain.
vAlthoughnumerousdiverseaminoacidsequencescanfunctionasactivationdomains,manyactivationdomainshaveanunusuallyhighpercentageofparticularaminoacids.
vGal4,Gcn4,andmostotheryeasttranscriptionfactorshaveactivationdomainsthatarerichinacidicaminoacids(asparticandglutamicacids).Theseso-calledacidicactivationdomainsgenerallyarecapableofstimulatingtranscriptioninnearlyalltypesofeukaryoticcells—fungal,animal,andplantcells.
vActivationdomainsfromsomeDrosophilaandmammaliantranscriptionfactorsareglutaminerich,andsomeareprolinerich;
vOthersarerichinthecloselyrelatedaminoacidsserineandthreonine,bothofwhichhavehydroxylgroups.
vHowever,somestrongactivationdomainsarenotparticularlyrichinanyspecificaminoacid.
MultiproteinComplexesFormonEnhancers
vModeloftheenhancesomethatformsontheβ-interferonenhancer.HeterodimericcJun/ATF-2,IRF-3,IRF-7,andNF-κB(aheterodimerofp50andp65)bindtothefourcontrolelementsinthe≈70-bpenhancer.CooperativebindingofthesetranscriptionfactorsisfacilitatedbyHMGI,whichbindstotheminorgrooveofDNA.ThecJun,ATF-2,p50,andp65proteinsallappeartointeractdirectlywithanHMGIboundadjacenttothem.BendingoftheenhancersequenceresultingfromHMGIbindingiscriticaltoformationofanenhancesome.DifferentDNA-bendingproteinsactsimilarlyatotherenhancers.
Example:
YeastActivatorGAL4
vRegulatesgenesinvolvedingalactosecatabolisminyeast
vGAL4bindsayeastDNAEnhancer:
UASG(UpstreamActivatingSequence)
vGAL4hasthreefunctions:
bindsUASG;activatestranscription;bindsGAL80,anotherregulatoryprotein。
vDNAbindinganddimerization
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