分子生物學(xué)-mbg-15gene regulation in prokaryotesRNA聚合酶和啟動(dòng)子互作σ因子

RegulationofGeneexpressioninprokaryoticcells概轉(zhuǎn)錄水平的調(diào)控RNA聚合酶和啟動(dòng)子的互作:σ因子lacoperon,trp轉(zhuǎn)錄的翻譯調(diào)控-衰減子系統(tǒng):trp翻譯水平的調(diào)DNA水平的調(diào)表達(dá)由調(diào)控蛋白控調(diào)控蛋白通過協(xié)同結(jié)合進(jìn)行轉(zhuǎn)錄調(diào)調(diào)控蛋白通過變構(gòu)作用進(jìn)行轉(zhuǎn)錄調(diào)調(diào)控并不局限于轉(zhuǎn)錄起始原核生物與真核生表達(dá)調(diào)控機(jī)制具有驚人的相似共同 與共同的分子基調(diào)控機(jī)理核酸分子調(diào)控機(jī)理核酸與蛋白質(zhì)分子間的互蛋白質(zhì)分子間的互調(diào)控層次調(diào)控層次Post-translationalGeneRegulationinInbacteriathemostcommonformofRNApolymeraserecognizespromotersformedfromthreeelements—the"-10","-35",and"UP"elements—andthestrengthofanygivenpromoterisdeterminedbywhichoftheseelementsitpossessesandhowwelltheymatchoptimum"consensus"sequences.Intheabsenceofregulatoryproteins,theseelementsdeterminetheefficiencywithwhichpolymerasebindstothepromoterand,oncebound,howreadilyitinitiatestranscription.PRINCIPLESOFTRANSCRIPTIONALGeneExpressionIsControlledbyRegulatoryInbacteria,genesareveryoftencontrolledbyextracellularthistypicallymeansmoleculespresentinthegrowthThesesignalsarecommunicatedtogenesbyregulatorywhichcomeintwopositiveregulators,oractivators;andnegativeregulators,orrepressors.TypicallytheseregulatorsareDNA-bindingproteinsthatrecognizespecificsitesatorneart estheycontrol.Anactivatorincreasestranscriptionoftheregulatedgene;repressorsdecreaseoreliminatethattranscription.Geneexpressioncanbecontrolledatanyofseveralstages,whichwedividebroadlyintotranscriptionalregulation,andpost-transcriptionalregulation.轉(zhuǎn)錄水平上的調(diào)控(transcriptionalregulation)Transcriptionofteniscontrolledatthestageofinitiation，maybecontrolledattermination…mRNA(differentialprocessingofRNA(differentialtranslationof原核與真核生物轉(zhuǎn)錄與翻譯的原核轉(zhuǎn)錄與翻譯相偶coupledtranscriptionand真核:初級轉(zhuǎn)錄本被加工剪接(splicing),轉(zhuǎn)運(yùn)至核外翻譯不同的生物使用不同的信號來指 調(diào)控原核生物中,營養(yǎng)狀況(nutritionalstatus)和環(huán)境因素(environmentalfactor)對 level)和發(fā)育階段(developmentalstage)是 表達(dá)由調(diào)控蛋白控結(jié) 和調(diào)AstructuralgenecodesforanyRNAorproteinproductotherthanaregulator.Aregulatorgenecodesforaproduct(typicallyprotein)thatcontrolstheexpressionofothergenes(usuallyattheleveloftranscription).正調(diào)控和負(fù)調(diào)InnegativeregulationarepressorproteinbindstoanoperatortopreventagenefrombeingInpositiveregulationanactivatorisrequiredtobindatthepromoterinordertoenableRNApolymerasetoinitiatetranscription.在沒有調(diào)節(jié)蛋白質(zhì)與目標(biāo)序列結(jié)合時(shí)，表達(dá)；當(dāng)調(diào)節(jié)蛋白質(zhì)與目標(biāo)序列結(jié)合時(shí)，表達(dá)被關(guān)閉，這種控制系統(tǒng)叫做負(fù)調(diào)控(negativeregulation。負(fù)調(diào)控中的調(diào)節(jié)蛋白質(zhì)稱在沒有調(diào)節(jié)蛋白質(zhì)與目標(biāo)序列結(jié)合時(shí)，是關(guān)閉的；當(dāng)調(diào)節(jié)蛋白質(zhì)與目標(biāo)序列結(jié)合時(shí)，就被開啟，這種控制系統(tǒng)叫做正調(diào)控(positiveregulation)。正調(diào)控中的調(diào)節(jié)蛋白質(zhì)RegulationcanbenegativeorGeneactivityisregulatedbythespecificinteractionsofthetrans-actingproducts(usuallyproteins)withthecis-actingsequences(usuallysitesinDNA).順式作用和反式作Atrans-actingproductcanfunctiononanycopyofitsDNA.ThisimpliesthatitisadiffusibleproteinAcis-actingsiteaffectstheactivityonlyofsequencesonitsownmoleculeofDNA(orRNA);thispropertyusuallyimpliesthatthesitedoesnotcodeforprotein. 白特異性識別和結(jié)合的特異DNA序列。包括啟動(dòng)子、上游啟動(dòng)子元件、反式作用因子：是指真核細(xì)胞內(nèi)含有的大量可以通過直接或間接結(jié)合式作用元件而調(diào) 轉(zhuǎn)錄活性的蛋白質(zhì)因子Innegativecontrol,atrans-actingrepressorbindstothecis-actingoperatortoturnoffInpositivecontrol,trans-actingfactorsmustbindtocis-actingsitesinorderforRNApolymerasetoinitiatetranscriptionatthe調(diào)控蛋白(regulatoryproteins)通過協(xié)同結(jié)合Intheabsenceofregulatoryproteins,RNAisoftenexpressedatalow,basalBasalConstitutiveActivationbyrecruitmentofRNApolymerase.(a)Intheabsenceofbothactivatorandrepressor,RNApolymeraseoccasionallybindsthepromoterspontaneouslyandinitiatesalowlevel(basallevel)oftranscription,(b)BindingoftherepressortotheoperatorsequenceblocksbindingofRNApolymeraseandsoinhibitstranscription,(c)RecruitmentofRNApolymerasebytheactivatorgiveshighlevelsoftranscription.RNApolymeraseisshownrecruitedintheclosedcomplex.Itthenspontaneouslyisomerizestotheopencomplexandinitiatestranscription.Ifboththerepressorandactivatorarepresentandfunctional,theactionoftherepressortypically thatoftheactivator.(敗事容易，成事難！Thiscaseisnotshowninthefigure)Arepressorisaproteinthatinhibitsexpressionofagene.ItmayacttopreventtranscriptionbybindingtoanoperatorsiteinDNA,ortopreventtranslationbybindingtoRNA.Recruitment(募集）amechanismofactivationinwhichtheactivatorsimultaneouslybindstoDNAandRNApolymersase,andholdstheRNApolymeraseinplacetoinitiatetranscription.調(diào)控蛋白通過變構(gòu)作用進(jìn)行轉(zhuǎn)錄調(diào)Allostery(變構(gòu)）:changeintheconformationofaproteinornucleicacidbroughtaboutbybindingofanothersubstanceatasiteotherthantheactiveSomeActivatorsWorkbyAllosteryandRegulateafterRNAPolymeraseInsomecases,RNApolymerasebindsefficientlyunaidedandformsastableclosedcomplex.Butthatclosedcomplexdoesnotspontaneouslyundergotransitiontotheopencomplex.Atthispromoter,anactivatormuststimulatethetransitionfromclosedtoopencomplex,sincethattransitionistheraimitingActivatorsthatstimulatethiskindofpromoterworkbytriggeringaconformationalchangeineitherRNApolymeraseorDNA.Thatis,theyinteractwiththestableclosedcomplexandinduceaconformationalchangethatcausestransitiontotheopencomplex.Thismechanismisanexampleofallostery.AllostericactivationofRNApolymerase.(a)BindingofRNApolymerasetothepromoterinastableclosedcomplex,(b)TheactivatorinteractswithpolymerasetotriggertransitiontotheopencomplexandhighlevelsofDNA-bindingproteinsthatinteractwitheachotheroftenbindtoadjacentsites.ButsomeproteinsinteractwitheachotherevenwhenboundtositeswellseparatedontheDNA.To modatethisinteraction,theDNAbetweenthesitesloopsout,bringingthesitesintoproximitywithoneanother.InteractionsproteinsboundtoCooperativebindingproteinstoadjacentCooperativebindingofproteinstoseparatedDNA-bendingproteincanfacilitateinteractionbetweenDNA-bindingproteins.AproteinthatbendsDNAbindstoasitebetweentheactivatorbindingsiteandthepromoter.ThisbringsthetwositesclosertogetherinspaceandtherebyhelpstheinteractionbetweentheDNA-boundactivatorandpolymerase.調(diào)控并不局限于轉(zhuǎn)錄起始RegulationofGeneexpressioninprocaryoticcells概轉(zhuǎn)錄水平的調(diào)控RNA聚合酶和啟動(dòng)子的互作:σ因子lacoperon,trp轉(zhuǎn)錄的翻譯調(diào)控-衰減子系統(tǒng):trp翻譯水平的調(diào)DNA水平的調(diào)RNA聚合酶和啟動(dòng)子的互作:σ因E.coliusesalternativesigmafactorstorespondtogeneralenvironmentalchanges.Inadditiontoσ70,E.colihasseveralsigmafactorsthatareinducedbyparticularenvironmentalconditions.E.colisigmafactorsrecognizepromotersdifferentconsensus轉(zhuǎn)錄起始的調(diào)控 Clusteringofstructuralgenesallowsthemtobe ycontrolledbymeansofinteractionsatasinglepromoter:asaresultoftheseinteractions,theentiresetofgenesiseithertranscribedornotAnoperonisaunitofbacterialgeneexpressionandregulation,includingstructuralgenesandcontrolelementsinDNArecognizedbyregulatorgeneproduct(s).JacobandMonodin酶的誘導(dǎo)現(xiàn)象個(gè)分子/細(xì)胞；有乳糖時(shí)，在2-3分鐘內(nèi)開始出現(xiàn)β–半乳糖苷酶，很快達(dá)到5000 子模型成功地解釋了β–半乳糖苷酶誘導(dǎo)合成的自1961年至今，對乳糖子不斷深入地研究已形成一套完整的調(diào)控機(jī)理的體系。為原核生物表達(dá)調(diào)控的研究細(xì)胞中的酶有的可以被誘導(dǎo)有的則可以被阻遏如E.colilacOperonNegativecontrol(byPositivecontrol(byActivatorandrepressorfunctionlacTheclusterofthethreelacstructuralgenes,Thelacoperonoccupies~6000bpofDNA.Attheleft,thelacIgenehasitsownpromoterandterminator. ofthelacIregionisadjacenttothepromoter(P).Theoperator(O),occupiesthefirst26bpofthetranscriptionunit.ThelonglacZgenestartsatbase39,andisfollowedbythelacYandlacAgenesandaterminator.主要有結(jié)構(gòu)區(qū)和上游的控制區(qū)，此外上游的一個(gè)調(diào)lacI控制Promoter（P） 結(jié)構(gòu) β- Thethreelacgenes—lacZ,lacY,andlacA—arearrangedontheE.coligenomeandarecalledthelacThelacpromoter,locatedatthe5'endoflacZ,directstranscriptionofallthreegenesasasinglemRNA(calledapolycistronicmessagebecauseitincludesmorethanonegene);thismRNAistranslatedtogivethethreeproteinproducts.ThelacZgeneencodestheenzymeβ-galactosidase半乳糖苷酶,whichcleavesthesugarlactoseintogalactose半乳andglucosebothofwhichareusedbythecellasenergysources.ThelacYgeneencodesthelactosepermease,aproteinthatintothecellmembraneandtransportslactoseintotheThelacAgeneencodesthiogalactoside[θai?u'g?l?kt?said硫代半乳糖苷transacetylase[tr?nz?'setileis轉(zhuǎn)乙酰酶,whichridsthecelloftoxicthiogalactosidesthatalsogettransportedinbylacY.Thelacoperon:Thethreegenes(lacZ,Y,andA)aretranscribedasasinglemRNAfromthepromoter(asindicatedbythearrow).TheCAPsiteandtheoperatorareeachabout20bp.TheoperatorlieswithintheregionboundbyRNApolymeraseatthepromoter,andtheCAPsi iesjustupstreamofthepromoter.Thesegenesareexpressedathighlevelsonlywhenlactoseisavailable,andglucose—thepreferredenergysource—isnot.Tworegulatoryproteinsareinvolved:oneisanactivatorcalledCAP,theotherarepressorcalledtheLacThenameCAPstandsforCataboliteActivatorProtein分解代謝物激活蛋白),butthisactivatorisalsoknownasCRP(forcAMPReceptorProtein環(huán)腺苷酸受體蛋白).LacrepressorcanbindDNAandrepresstranscriptiononlyintheabsenceoflactose.cataboliterepression分解代謝物阻Inthepresenceoflactose,therepressorisinactive esde-repressedCAPcanbindDNAandactivatethelacgenesonlytheabsenceofThus,thecombinedeffectofthesetworegulatorsensuresthatt esareexpressedatsignificantlevelsonlywhenlactoseispresentandglucoseabsent.Specificcontrolofthelacgenesdependsontheavailabilityofthesubstrateslactose/glucosetothebacteriumThefirstcontrolmechanismistheregulatoryresponsetolactose,whichusesanintracellularregulatoryproteincalledthelactoserepressortohinderproductionofβ-galactosidaseintheabsenceoflactose.Thesecondcontrolmechanismisaresponsetoglucose,whichusesthecataboliteactivatorprotein(CAP)togreatlyincreaseproductionofβ-galactosidaseintheabsenceofglucose.CyclicadenosinemonophosphatecAMPisasignalmoleculewhoseprevalenceisinversely(相反地）proportionaltothatofglucose.ItbindstotheCAP,whichinturnallowstheCAPtobindtotheCAPbindingsite(a16bpDNAsequenceupstreamofthepromoter),whichassiststheRNApolymeraseinbindingtotheDNA.Intheabsenceofglucose,thecAMPconcentrationishighandbindingofCAP-cAMPtotheDNAsignificantlyincreasestheproductionofβ-galactosidase,enablingthecelltohydrolyse(digest)lactoseandreleasegalactoseandglucose.lacOperonNegativecontrol(byPositivecontrol(byActivatorandrepressorfunction乳 子所受的負(fù)調(diào)控(negativeRepressorandRNApolymerasebindatsitesthatoverlapthetranscriptionstartpointofthelacAninducer(suchaslactose)isasmallmoleculethattriggersgenetranscriptionbybindingtoaregulatorprotein.AcorepressorisasmallmoleculethattriggersrepressiontranscriptionbybindingtoaregulatorIntheabsenceofaninducer,t esarenottranscribed,becauserepressorproteinisinanactiveformthatisboundtotheoperator.Whenaninducerisadded,therepressorisconvertedintoaninactiveformthatleavestheoperator.suchasAninducerfunctionsbyconvertingtherepressorproteinintoaninactiveform.Repressorhastwobindingsites,onefortheoperatorandanotherfortheinducer.Repressorisinactivatedbylosteric變構(gòu)的interactioninwhichbindingofinduceratitssitechangesthepropertiesoftheDNA-bindingsite.lacOperonNegativecontrol(byPositivecontrol(byActivatorandrepressorfunction乳 子所受的正調(diào)控(positive正調(diào)控系統(tǒng)中主要的作用因子是cAMP-CAP(CRP)，Cataboliterepression（分解代謝產(chǎn)物阻遏Cataboliteactivatorprotein(CAP，分解代謝產(chǎn)物激活蛋白)isactiveonlyinthepresenceofcyclicAMP.ByreducingthelevelofcyclicAMP,glucoseinhibitstranscriptionofoperonsthatrequireCRPCAP具有獨(dú)立的激活結(jié)構(gòu)域和DNA結(jié)合結(jié)構(gòu) CAPcouldinteractdirectlywithRNAItalsocouldactuponDNAtochangeitsstructureinsomewaythatassistsRNApolymerasetobind.CRPactivator(CAPactivator)isneededforRNApolymerasetoinitiatetranscriptionofmanyoperonsofE.coli.如：gal乳 子的負(fù)調(diào)控和正調(diào)負(fù)調(diào)控系統(tǒng)中主要的作用因子是阻遏蛋白，誘導(dǎo)正調(diào)控系統(tǒng)中主要的作用因子是cAMP-CAP，葡lCAP介導(dǎo)葡萄糖信lacrepressor介導(dǎo)乳糖信號Repressorandactivator(CAP)Expressionofthelacgenes:Thepresenceorabsenceofthesugarslactoseandglucosecontrolthelevelofexpressionofthelacgenes.Highlevelsofexpressionrequirethepresenceoflactose(andhencetheabsenceoffunctionalLacrepressor)andabsenceofthepreferredenergysource,glucose(andhencepresenceoftheactivatorCAP).Whenboundtotheoperator,LacrepressorexcludespolymerasewhetherornotactiveCAPispresent.CAPandLacrepressorareshownassingleunits,butCAPactuallybindsDNAasadimer,andLacrepressorbindsasatetramer.CAPrecruitspolymerasetothelacpromoterwhereitspontaneouslyundergoesisomerizationtotheopencomplex(thestateshowninthebottomline).lacOperonNegativecontrol(byPositivecontrol(byActivatorandrepressorfunctionActivator和repressor共同控制Thecontrolregionofthelacoperon.Thenucleotidesequenceandorganizationofthelacoperoncontrolregionareshown.ThecoloredbarsaboveandbelowtheDNAshowregionscoveredbyRNApolymeraseandtheregulatoryproteins.NotethatLacrepressorcoversmoreDNAthanthatsequencedefinedastheminimaloperatorbindingsite,andRNApolymerasemorethanthatdefinedbythesequencesthatmakeupthepromoter.Thelacoperatoroverlapsthepromoter,andsorepressorboundtotheoperatorphysicallypreventsRNApolymerasefrombindingtothepromoterandthusinitiatingRNAsynthesis.CAPandLacRepressorBindDNAUsingCommonStructuralRecognitionofspecificDNAsequencesisachievedusingaconservedregionofsecondarystructurecalledahelix-turn-helix. iscomposedoftwoαhelices,oneofwhich—therecognitionhelix—fitsintothemajorgrooveoftheDNA.ThecontactsmadebetweentheaminoacidsidechainsprotrudingfromtherecognitionhelixandtheedgesofthebasescanbemediatedbydirectH-bonds,indirectH-bonds(bridgedbywatermolecules),orVanderWaalsforces.Thesecondhelixofthehelix-turn-helix sitsacrossthemajorgrooveandmakescontactwiththeDNAbackbone,ensuringproperpresentationoftherecognitionhelix,andatthesametimeaddingbindingenergytotheoverallprotein-DNAinteraction.CAP和repressor以相同方式結(jié)合Bindingofaproteinwithahelix-turn-helix toDNA.Theprotein,asistypicallythecase,bindsasadimer,andthetwosubunitsareindicatedbytheshadedcircles.Thehelix-turn-helixmotifonea onomerisindicated;the"recognitionhelix"islabeledR.盡管Jacob&Monod的描述尚不完整，但他們關(guān)于反式調(diào)控識別蛋的調(diào)控的研究。只有葡萄糖時(shí)利用葡萄糖;只有乳糖時(shí),利用乳糖,SowhenE.colifindsbothglucoseandinthemedium,itmetabolizestheglucoseandrepressestheuseThephenomenonofglucoserepressionfollowsfromtheabilityofglucosetopreventtheuseofalternativecarbonsources.這一現(xiàn)象涉及到乳 子的正調(diào)控系統(tǒng)ThelacControlcircuitsareversatileandcanbedesignedtoallowpositiveornegativecontrolofinductionorThetrpRegulationofGeneexpressioninprocaryoticcells概轉(zhuǎn)錄水平的調(diào)控RNA聚合酶和啟動(dòng)子的互作:σ因子lacoperon,trp轉(zhuǎn)錄的翻譯調(diào)控-衰減子系統(tǒng):trp翻譯水平的調(diào)DNA水平的調(diào)大腸桿菌的色氨 子Thetrp被最終合成產(chǎn)物所阻遏，是一個(gè)典型的可阻 結(jié)結(jié)構(gòu) ：trpE，trpD，trpC，trpB，trpA，調(diào)控區(qū):promoter、operator、前導(dǎo)肽和弱化與trpO結(jié)合的阻遏蛋白由相距甚遠(yuǎn)的trpRThetrpoperonconsistsoffivecontiguousstructuralgenesprecededbyacontrolregionthatincludesapromoter,operator,leaderpeptidecodingregion,andattenuator.ControlofthetrpTryptophan’sroleinnegativecontrolofthetrpoperon.ControlofthetrpoperonbyAttenuationdescribestheregulationofbacterialoperonsbycontrollingterminationoftranscriptionatasi ocatedbeforethefirststructuralgene.Anattenuatorisaterminatorsequenceatwhichattenuationoccurs.TheE.colitryptophanoperoniscontrolledbyattenuationKeyAnattenuator(intrinsicterminator)islocatedbetweenthepromoterandthefirstgeneofthetrpcluster.Theabsenceoftryptophansuppressesterminationandresultsina10×increaseintranscription.Thetrpleaderregioncanexistinalternativebase-pairedconformations.Thecentershowsthefourregionsthatcanbasepair.Region1iscomplementarytoregion2,whichiscomplementarytoregion3,whichiscomplementarytoregion4.Ontheleftistheconformationproducedwhenregion1pairswithregion2,andregion3pairswithregion4.Ontherightistheconformationwhenregion2pairswithregion3,leavingregions1and4unpaired.AttenuationcanbecontrolledbyKeyTheleaderregionofthetrpoperonhasa14codonopenreadingframethatincludestwocodonsfortryptophan.ThestructureofRNAattheattenuatordependswhetherthisreadingframeisInthepresenceoftryptophan,theleaderistranslated,andtheattenuatorisabletoformthehairpinthatcausesIntheabsenceoftryptophan,theribosomestallsatthetryptophancodonsand ternativesecondarystructurepreventsformationofthehairpin,sothattranscriptionThealternativesforRNApolymeraseattheattenuatordependonthelocationoftheribosome,whichdetermineswhetherregions3and4canpairtoformtheterminatorhairpin.AnattenuatorcontrolstheprogressionofRNApolymeraseintothetrpgenes.RNApolymeraseinitiatesatthepromoterandthenproceedstoposition90,whereitpausesbeforeproceedingtotheattenuatoratposition140.Intheabsenceoftryptophan,thepolymerasecontinuesintothestructuralgenes(trpEstartsat+163).Inthepresenceoftryptophanthereis~90%probabilityofterminationtoreleasethe140-baseleaderRNA.RegulationofGeneexpressioninprocaryoticcells概轉(zhuǎn)錄水平的調(diào)控RNA聚合酶和啟動(dòng)子的互作:σ因子