Gene-X-ppt(Gene10-基因十)--Chapter19

Chapter19ProkaryoticTranscription19.1Introduction•Transcriptionis5′to3′onatemplatethatis3′to5′.•coding(nontemplate)strand–TheDNAstrandthathasthesamesequenceasthemRNAandisrelatedbythegeneticcodetotheproteinsequencethatitrepresents.•RNApolymerase–AnenzymethatsynthesizesRNAusingaDNAtemplate(formallydescribedasaDNA-dependentRNApolymerase).FIGURE01:OnestrandofDNAistranscribedintoRNA19.1Introduction•promoter–AregionofDNAwhereRNApolymerasebindstoinitiatetranscription.•startpoint–ThepositiononDNAcorrespondingtothefirstbaseincorporatedintoRNA.•terminator–AsequenceofDNAthatcausesRNApolymerasetoterminatetranscription.•transcriptionunit–ThesequencebetweensitesofinitiationandterminationbyRNApolymerase;itmayincludemorethanonegene.FIGURE02:Promotersandterminatorsdefinetheunit19.1Introduction•upstream–Sequencesintheoppositedirectionfromexpression.•downstream–Sequencesproceedingfartherinthedirectionofexpressionwithinthetranscriptionunit.•primarytranscript–TheoriginalunmodifiedRNAproductcorrespondingtoatranscriptionunit.19.2TranscriptionOccursbyBasePairingina“Bubble”ofUnpairedDNA•RNApolymeraseseparatesthetwostrandsofDNAinatransient“bubble”andusesonestrandasatemplatetodirectsynthesisofacomplementarysequenceofRNA.•Thebubbleis12to14bp,andtheRNA–DNAhybridwithinthebubbleis8to9bp.FIGURE03:RNAsynthesisoccursinthetranscriptionbubbleFIGURE05:RNApolymerasesurroundsthebubble19.3TheTranscriptionReactionHasThreeStages•RNApolymerasebindstoapromotersiteonDNAtoformaclosedcomplex.•RNApolymeraseinitiatestranscription(initiation)afteropeningtheDNAduplextoformatranscriptionbubble(theopencomplex).FIGURE06:RNApolymerasecatalyzestranscription19.3TheTranscriptionReactionHasThreeStages•DuringelongationthetranscriptionbubblemovesalongDNAandtheRNAchainisextendedinthe5′→3′directionbyaddingnucleotidestothe3′end.•Transcriptionstops(termination)andtheDNAduplexreformswhenRNApolymerasedissociatesataterminatorsite.19.4BacterialRNAPolymeraseConsistsofMultipleSubunits•holoenzyme–TheRNApolymeraseformthatiscompetenttoinitiatetranscription.Itconsistsofthefivesubunitsofthecoreenzymeandσfactor.•BacterialRNAcorepolymerasesare~400kDmultisubunitcomplexeswiththegeneralstructureα2ββ′.FIGURE07:RNApolymerasehas4typesofsubunit19.4BacterialRNAPolymeraseConsistsofMultipleSubunits•Catalysisderivesfromtheβandβ′subunits.•CTD(C-terminaldomain)–ThedomainofRNApolymerasethatisinvolvedinstimulatingtranscriptionbycontactwithregulatoryproteins.FIGURE08:TheupstreamfaceofthecoreRNApolymeraseAdaptedfromK.M.GeszvainandR.Landick(ed.N.P.Higgins).TheBacterialChromosome.AmericanSocietyforMicrobiology,2004.FIGURE09:ThestructureofRNApolymeraselookingthroughthemainchannelStructurefromProteinDataBank1HQM.L.Minakhin,etal.,Proc.Natl.Acad.Sci.USA98(2001):892-897.19.5RNAPolymeraseHoloenzymeConsistsoftheCoreEnzymeandSigmaFactor•BacterialRNApolymerasecanbedividedintotheα2ββ′coreenzymethatcatalyzestranscriptionandthesubunitthatisrequiredonlyforinitiation.•SigmafactorchangestheDNA-bindingpropertiesofRNApolymerasesothatitsaffinityforgeneralDNAisreducedanditsaffinityforpromotersisincreased.FIGURE10:Sigmafactorcontrolsspecificity19.6HowDoesRNAPolymeraseFindPromoterSequences?•TherateatwhichRNApolymerasebindstopromoterscanbetoofasttobeaccountedforbysimplediffusion.•RNApolymerasebindstorandomsitesonDNAandexchangesthemwithothersequencesuntilapromoterisfound.FIGURE11:ProposedmechanismsforhowRNApolymerasefindsapromoterAdaptedfromC.Bustamante,etal.,J.Biol.Chem.274(1999):166665-166668.19.7TheHoloenzymeGoesthroughTransitionsintheProcessofRecognizingandEscapingfromPromoters•WhenRNApolymerasebindstoapromoter,itseparatestheDNAstrandstoformatranscriptionbubbleandincorporatesnucleotidesintoRNA.FIGURE12:RNApolymerasepassesthroughseveralstepspriortoelongationAdaptedfromS.P.Haugen,W.Ross,andR.L.Gourse,Nat.Rev.Microbiol.6(2008):507-519.19.7TheHoloenzymeGoesthroughTransitionsintheProcessofRecognizingandEscapingfromPromoters•ternarycomplex–ThecomplexininitiationoftranscriptionthatconsistsofRNApolymeraseandDNAaswellasadinucleotidethatrepresentsthefirsttwobasesintheRNAproduct.•Theremaybeacycleofabortiveinitiationsbeforetheenzymemovestothenextphase.•SigmafactorisusuallyreleasedfromRNApolymerasewhenthenascentRNAchainreaches~10basesinlength.FIGURE13:RNApolymerasechangessizeatinitiation19.8SigmaFactorControlsBindingtoDNAbyRecognizingSpecificSequencesinPromoters•conservedsequence–Sequencesinwhichmanyexamplesofaparticularnucleicacidorproteinarecomparedandthesameindividualbasesoraminoacidsarealwaysfoundatparticularlocations.•Apromoterisdefinedbythepresenceofshortconsensussequencesatspecificlocations.19.8SigmaFactorControlsBindingtoDNAbyRecognizingSpecificSequencesinPromoters•Thepromoterconsensussequencesusuallyconsistofapurineatthestartpoint,ahexamerwithasequenceclosetoTATAATcenteredat~–10(–10elementorTATAbox),andanotherhexamerwithasequencesimilartoTTGACAcenteredat~–35(–35element).•Individualpromotersusuallydifferfromtheconsensusatoneormorepositions.19.8SigmaFactorControlsBindingtoDNAbyRecognizingSpecificSequencesinPromoters•Promote