K原核生物的转录

K原核生物的转录第1页/共54页Transcription

是指以adouble-strandedDNA为模板合成asingle-strandedRNA.

RNAsynthesisoccursinthe5’3’directionanditssequencecorrespondstothatoftheDNAstrandwhichisknownasthesensestrand（有义链）.ThetemplateofRNAsynthesisistheantisensestrand.Necessarycomponents:promoter/template,RNApolymerase,NTPs,terminator/template第2页/共54页(-)strandisantisensestrand.(+)strandissensestrand第3页/共54页K1

转录的基本原则

Initiation:polymeraseandpromoters

Elongation:RNApolymerase

Termination:terminator第4页/共54页ATACGTATGC+1promoterterminatorTranscribedregionRNADNATranscriptionAntisensestrandAUACGStructureofatranscriptionunit第5页/共54页Whatisapromoter(启动子)?ThesequenceofDNAneededforRNApolymerasetobindtothetemplateandaccomplishtheinitiationreaction位于蛋白质编码区的上游（5’端）含有短而保守的DNA序列，不同基因的启动子中序列经常是保守的。第6页/共54页InitiationBindingofanRNApolymerasetothedsDNA（非专一不稳定的复合物在模板移动）Slidetofindthepromoter（封闭的“酶-启动子”二元复合物）UnwindtheDNAhelix

（开放的“酶-启动子”二元复合物）SynthesisoftheRNAstrandatthestartsite(initiationsite),thispositioncalledposition+1

（“酶-启动子-rNTP”三元复合物）第7页/共54页Transcriptionbubble第8页/共54页ElongationRNApolymeraseadds核糖核苷酸tothe3’-endand延伸thegrowingRNAchaininthedirectionof5’3’（E.coli:40nt/sec)酶自身沿着反义DNA链（模板）的3’5’方向移动。酶移动时局部解旋，经过后重新形成双螺旋。第9页/共54页Elongation第10页/共54页Termination转录复合物的解体andseparationofRNAstrandfromDNAOccurringattheterminator(often茎环或发夹二级structure),someneedrhoproteinasaccessoryfactor.第11页/共54页RNAhairpin(发卡)structure5‘NNNNAAGCGCCGNNNNCCGGCGCUUUUUU-OHNNNNCG•CC•GC•GG•CC•GG•CA•UA•U…NNNNUUUU-OH第12页/共54页StepsforRNAtranscription第13页/共54页2.

RequiresDNAforactivityandismostactivewithadouble-strandedDNAastemplate.5’3’synthesis(NMP)n+NTP(NMP)n+1+PPiRNApolymerase:synthesisofRNAstrandfromDNAtemplate.1.

Requiresnoprimerforpolymerization3.RequireMg2+forRNAsynthesisactivity第14页/共54页4.AllRNApolymeraseslack3’5’exonucleaseactivity,andoneerrorusuallyoccurswhen104to105nucleotidesareincorporated.5.usuallyaremultisubunitenzyme,butnotalways.6.Differentfromorganismtoorganism7.E.colihasasingleDNA-directedRNApolymerasethatsynthesizesalltypesofRNA.第15页/共54页K2E.coliRNApolymeraseBoth起始&延伸起始only36.5KD36.5KD151KD155KD11KD70KD465kd第16页/共54页RNAsynthesisrate:40ntpersecondat37oC

非球形结构，圆柱形孔道旁有一突起，孔道大小表明theenzymecomplexcanbinddirectlyto16bpofDNA.Thewholepolymerasebindsover60bp.第17页/共54页E.coliRNApolymerase:全酶:2’forinitiation核心酶:2’forelongation第18页/共54页RNApol亚基第19页/共54页E.colipolymerase:asubunitTwoidenticalsubunitsinthecoreenzymeEncodedbytherpoAgeneRequiredforcoreproteinassemblyMayplayaroleinpromoterrecognition

第20页/共54页EncodedbyrpoBgene.ThecatalyticcenteroftheRNApolymeraseRifampicin(利福平)：bindtotheβsubunit,andinhibittranscriptioninitiation.MutationinrpoBgenecanresultinrifampicinresistance.阻止起始但不影响延伸，治疗G+的感染和结核病。Streptolydigins(利迪链菌素)：resistantmutationsaremappedtorpoBgeneaswell.Inhibitstranscriptionelongationbutnotinitiation.3. bsubunitmaycontaintwodomainsresponsiblefor转录起始和延伸。E.colipolymerase:bsubunit第21页/共54页EncodedbytherpoCgene.BindstwoZn2+ionsandmayparticipateinthecatalyticfunctionofthepolymeraseHeparin(肝素)：bindstotheb’subunit体外抑制转录.HeparincompeteswithDNAforbindingtothepolymerase.3. b’subunitmayberesponsibleforbindingtothetemplateDNA

（与模板结合有关）.E.colipolymerase:b’subunit第22页/共54页Many原核生物containmultiplesfactorstorecognizedifferentpromoters.ThemostcommonsfactorinE.coliiss70.BindingofthesfactorconvertsthecoreRNApolintotheholoenzyme.E.colipolymerase:sfactor第23页/共54页sfactoriscriticalinpromoterrecognition,bydecreasing核心酶对非特异序列的亲和力(104)andincreasingtheaffinityforthecorrespondingpromotersfactorisreleasedfromtheRNApolafterinitiation(RNAchainis8-9nt)sfactor数目明显少于theothersubunitsoftheRNApol（30%）.E.colipolymerase:sfactor第24页/共54页K3TheE.coliσ70promoterPromotersequences:

-10sequenceand-35sequence2. Transcriptionstartsite3. Promoterefficiency第25页/共54页Promoters:含有RNApol特异性结合和转录起始所需的保守序列。ATACGTATGC+1promoterterminatorTranscribedregionRNADNATranscriptionAntisensestrandDifferentpromotersresultindifferingefficienciesoftranscriptioninitiation,whichinturnregulatetranscription.AUACG第26页/共54页Promotersequence位于thestartsiteoftranscription(position+1)上游,一般为负数ContainsshortconservedsequencescriticalforspecificbindingofRNApolymeraseandtranscriptioninitiation验证主要通过mutationsthatenhanceordiminishtherateoftranscriptionofgene第27页/共54页第28页/共54页s70promoterConsistsofasequenceofbetween40and60bp-55to+20:boundbythepolymerase-20to+20:tightlyassociatedwiththepolymeraseandprotectedfromnucleasedigestionbyDNaseΙ直至–40区域对于启动子功能是必须的-10and–35sequence:particularlyimportantforpromoterfunction---5-8bp---GCTATTGACATATAAT-----16-18bp-------+1-35sequence-10sequence第29页/共54页-10sequence(Pribnowbox)6bpsequencewhichiscenteredataroundthe–10position(Pribnow,1975).AconsensussequenceofTATAATThefirsttwobases(TA)andthefinalTaremosthighlyconservedamongotherE.colipromoters六聚体距离转录起始位点5to8bp-10序列似乎是聚合酶启动DNA解旋的序列第30页/共54页-35sequence组成增强与聚合酶因子相识别和相互作用的识别区Aconserved六聚体序列aroundposition–35AconsensussequenceofTTGACAThefirstthreepositions(TTG)arethemostconservedamongE.colipromoters.Separatedby16-18bpfromthe–10boxin90%ofallpromoters第31页/共54页TranscriptionstartsiteApurinein90%ofallgenesGismorecommonatposition+1thanAOften,起始位点两侧为CandT(i.e.CGTorCAT)Thesequencearoundthestartsiteinfluencesinitiation第32页/共54页ThesequencesoffiveE.colipromotersConsensusTTGACATATAAT第33页/共54页Promoterefficiency(1)不同启动子与不同基因的转录速率千差万别varybyupto1000-fold.The–35sequence,-10sequence,and起始位点附近序列allinfluenceinitiationefficiency.最初转录30bases控制theRNApolymerase离开thepromoter,henceinfluencestherateofthetranscriptionandtheoverallpromoterstrength.第34页/共54页Promoterefficiency(2)DNA模板的负超螺旋可增强转录起始Somepromotersequencearenotstrongenoughtoinitiatetranscriptionundernormalcondition,activatingfactorisrequiredforinitiation.Forexample,LacpromoterPlacrequirescAMPreceptorprotein(CRP)第35页/共54页K4转录的起始、延伸与终止PromoterbindingDNAunwinding

RNAchaininitiationRNAchainelongationRNAchainterminationRho-dependenttermination第36页/共54页Binding第37页/共54页UnwindingInitiationElongationTermination第38页/共54页Promoter结合Thecoreenzyme(2’)

hasnonspecificDNAbinding(loosebinding,全酶20000foldless).ThefactorenhancesthespecificityofthecoreRNApolymerase(2’)

forpromoterbinding(100x)（全酶结合到正确的位点）Thepolymerasefindsthepromoter–35and–10sequencesbyslidingalongtheDNAextremelyrapidlyandformingaclosedcomplexwiththepromoterDNA(Theinitialcomplexofthepolymerasewiththebase-pairedpromoterDNA)第39页/共54页DNA解旋

NecessarytounwindtheDNAsothattheantisensestrandtobecomeaccessibleforbasepairing,carriedoutbythepolymerase.

负超螺旋能增进许多基因的转录butnotallfacilitatingbyunwinding.TheinitialunwindingoftheDNAresultsinformationofanopencomplexwiththepolymeraseandthisprocessisreferredtoastightbinding

第40页/共54页RNAchain起始NoprimerisneededStartwithaGTP(morecommon)orATPInitially掺入first2nucleotides，thefirst9ntareincorporatedwithoutpolymerasemovementalongtheDNAorσfactorrelease（未清除启动子）abortiveinitiations,whichareimportantfortheoverallrateoftranscription，对于聚合酶要花多长时间离开启动子并允许另一个聚合酶起始新一轮转录重要作用Theminimumtimeforpromoterclearanceis1-2seconds(arelativelylongevent)第41页/共54页第42页/共54页RNAchain延伸σFactorisreleasedtoformaternarycomplexofthepol-DNA-RNA(newlysynthesized),causingthepolymerasetoprogressalongtheDNA(promoterclearance)Transcriptionbubble(unwoundDNAregion,~17bp)movesalongtheDNAwithRNApolymerasewhichunwindsDNAatthefrontandrewindsitattherear3’partofRNAformshybridhelix(ca.12bp)withantisenseDNAstrand.TheE.colipolymerasemovesatanaveragerateof~40ntpersec,dependingonthelocalDNAsequence.第43页/共54页antisense第44页/共54页第45页/共54页RNAchain终止Termination:dissociationofRNA>re-annealingofDNA>releaseofRNApolTerminatorsequence终止序列(stopsignal停止信号):RNAhairpinverycommonAccessoryrhoprotein第46页/共54页RNAhairpin

1.RNA转录物是自身互补的2.GC-richfavouringthebasepairingstabilityandcausingthepolymerasetopause3.FollowedbyastretchoffourormoreUswhichresultinweakRNA-antisenseDNAstrandbinding，有利于解离第47页/