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李希分子医学教育部重点实验室lixi@TranscriptionandPost-transcriptionModification销售信

CentraldogmaWHICHCAMEFIRST,THEchickenortheegg?Thebiologicalsilenceshaveavariation:whichcamefirst,DNAorprotein?Yousee,amongthemanytasksperformedbyproteinsisassemblingDNAmolecules.ButDNAcontainstheinformationneededtomakeproteins.Sowhichcamefirst?RNAandRNAworldWalterGilbert1980NobelPrize

Origin-of-Life

TheoriesRNAhastheabilitytoactasbothgenesandenzymes

ThesynthesisofRNAmoleculesusingDNAstrandsasthetemplatessothatthegeneticinformationcanbetransferredfromDNAtoRNA.Fourstages:Initiation,Elongation,Termination,Post-transcriptionalmodification

Transcription

Onlythetemplatestrandisusedforthetranscription,butthecodingstrandisnot.Bothstrandscanbeusedasthetemplates.Thetranscriptiondirectionondifferentstrandsisopposite.Thisfeatureisreferredtoastheasymmetrictranscription.AsymmetrictranscriptionTemplateThetemplatestrandisthestrandfromwhichtheRNAisactuallytranscribed.Itisalsotermedasantisensestrand.Thecodingstrandisthestrandwhosebasesequencespecifiestheaminoacidsequenceoftheencodedprotein.Therefore,itisalsocalledassensestrand.BothprocessesuseDNAasthetemplate.Phosphodiesterbondsareformedinbothcases.Bothsynthesisdirectionsarefrom5´to3´.Similaritybetween

replicationandtranscription

ReplicationTranscriptionTemplateDoublestrandsSinglestrandSubstratedNTPNTPPrimeryesnoEnzymeDNApolymeraseRNApolymeraseProductdsDNAssRNABasepairA-T,G-CA-U,T-A,G-CDifferencesbetween

replicationandtranscription

ThewholegenomeofDNAneedstobereplicated,butonlysmallportionofgenomeistranscribedinresponsetothedevelopmentrequirement,physiologicalneedandenvironmentalchanges.DNAregionsthatcanbetranscribedintoRNAarecalledstructuralgenes.WhatdothemostDNAdoindeed?

GeneralconceptsofTranscriptionProcessThreephases:initiation,elongation,andtermination.TheprokaryoticRNA-polcanbindtotheDNAtemplatedirectlyinthetranscriptionprocess.TheeukaryoticRNA-polrequiresco-factorstobindtotheDNAtemplatetogetherinthetranscriptionprocess.

TranscriptionbubbleTranscriptioninprokaryotesTranscriptionUnit

OptimalPromoterThe–35sequenceisusedforinitialrecognition,andthe–10sequenceisusedforthemeltingreactionthatconvertsaclosedcomplextoanopencomplex.

Duringtranscription,thebubbleismaintainedwithinbacterialRNApolymerase,whichunwindsandrewindsDNA,maintainstheconditionsofthepartnerandtemplateDNAstrands,andsynthesizesRNA.

BacterialRNAPolymerasesAsingletypeofRNApolymeraseisresponsibleforalmostallsynthesisofmRNA,rRNAandtRNAinaeubacterium.About7,000

RNApolymerasemoleculesarepresentinanE.colicell.Probably2,000~5,000enzymesaresynthesizingRNAatanyonetime,thenumberdependingonthegrowthconditions.HowdoesRNApolymerasework?RNApol

bsubunitisthetargetofrifamycinHowmany

sigmafactorsexistinE.coli?

Howdoestranscriptioninitiate?FourstagesofTranscriptionTermination

Theterminatorisinthetranscript,nottheDNAFormsahairpinSelf-complementaryThehairpinstructureisthesignalforterminationRho(ρ)-dependentvs.ρ-independentIntrinsicterminatorsρ-independentAninvertedrepeatthatallowsahairpintoformattheendofthetranscriptsAstringofT’sinthenontemplatestrandthatresultsinastringofweakrU-dAbasepairsholdingthetranscripttothetemplatestrandRhofactorpursuesRNApolymerasealongtheRNAandcancauseterminationwhenitcatchestheenzymepausingatarho-dependentterminator.Terminationofρ-dependentTranscriptioninEukaryotesRNApolymerasesinEukaryotesRNApolymeraseItranscribesrRNA

RNApolymeraseIItranscribeshnRNARNApolymeraseIIItranscribestRNAandothersmallRNAs.Amanitaphalloides

(thedeathcap)Structureofα-amanitinAnimalRNAPolymerasesAnimalDNA-dependentRNAPolymerasesClassα-amanitinsensitivityMajorProducts

IInsensitiverRNA

IILowConc.(1-10nM)hnRNA

IIIHighconc.tRNA,5SRNAandsmallRNAs

Allhaveincommon2largesubunitsandanumberofsmallersubunits,aswellasbeingzincmetalloenzymes.

EukaryoticTranscriptionInitiationTranscriptioninitiationneedspromoterandupstreamregulatoryregions.The

cis-actingelements

arethespecificsequencesontheDNAtemplatethatregulatethetranscriptionofoneormoregenes.

Cis-actingelementRNA-poldoesnotbindthepromoterdirectly.RNA-polIIassociateswithsixtranscriptionfactors,TFIIA-TFIIH.Thetrans-actingfactorsaretheproteinsthatrecognizeandbinddirectlyorindirectlycis-actingelementsandregulateitsactivity.Transcriptionfactors

InitiationofRNApolymeraseIITATAboxisaseptamer(TATAAAA)at-25andisinvolvedinpositioningtheenzymeforcorrectinitiation.CAATbox(CCAATCT)isat–75andisrecognizedbyalargegroupoftranscriptionfactorsandplaysastrongroleindeterminingtheefficiencyofthepromoter.GCboxisat-90containsthesequenceGGGCGGandisrecognizedbythefactorSP1.ElementscombinationintypeIIPromoters

Enhancer;Dehancer;Silencer;UpstreamActivatingSequences(UAS)EnhancerEnhancersWorkUpstream,DownstreamorintheMiddleofaGeneTheyalsoworkforwardsorbackwardsPossiblewaysofworkingDifferenttranscriptionfactorsOrderofbinding(differingconcentrations)AffinityoftranscriptionfactorsTFIIAactivatesTBPbyrelievingtherepressionthatiscausedbytheTAFs

TFIIBbindsadjacenttoTBPandTATAboxTFIIDisacomplexproteincontainingaTATA-boxbindingproteinand8-10TBP-associatedfactors(TAFs)TBP:

TATA-bindingprotein

TAFs:

TBP-associatedfactorsTFIIFconsistsoftwosubunits.ThelargersubunithasanATP-dependentDNAhelicaseactivityandthesmallonecontactsthecorepolymerase.

TFIIEandTFIIHarerequiredforpromoterclearancetoallowRNApolymerasetocommencemovementawayfromthepromoter.ClassIItranscriptionfactorspolⅡTFⅡHTAFTFⅡFTAFTAFTFⅡATFⅡBTBPRNApolⅡwithtranscriptionfactorsformtranscriptioninitiationcomplex.TFIIDistheonlyfactorwhichcanrecognizespecificsites.TATADNATATAPOL-ⅡTFⅡFⅡAⅡBPreinitiationcomplexPOL-ⅡTFⅡFⅡHⅡETBPTAFTFⅡD-ⅡA-ⅡB-DNAcomplexⅡAⅡBTATAⅡHⅡECTD-PCTDtailofRNApolIIisphosphorylatedbyTFⅡHTBPTAFTBPTAFTBPTAFTBPCTD（CarboxylTerminalDomain)isrepeatedsequenceofTyr-Ser-Pro-Thr-Ser-Pro-Ser

TFIIHhasseveralactivities,includinganATPase,ahelicase,andakinaseactivitythatcanphosphorylatetheCTDtailofRNApolymeraseII;itisalsoinvolvedinrepairofdamagetoDNA.MostoftheTFIIfactorsarereleasedbeforeRNApolymeraseIIleavesthepromoter.PhosphorylationoftheCTDbythekinaseactivityofTFIIHmaybeneededtoreleaseRNApolymerasetostarttranscription.EndofInitiation

TranscriptionunitforRNApolymeraseIPromotersintypeIIIgeneupstreampromoter(type3)andinternalpromoter(type1,2)upstreampromoter:U6snRNAInternalpromoter:5SRNAandtRNAInitiationintypeIIIgenewithpolymeraseIIItRNA5SRNA

InitiationRNApolIRNApolIIIRNApolII__________________________________________________ATPrequirementnonoyes__________________________________________________AandBorTATAboxcoreconsensussq.coreelementCboxInr

__________________________________________________CAATboxupstreamelementUCEGCboxetc__________________________________________________generalTFsSL1TFIIIABCvariousTFIIs___________________________________________________upstreamfactorsUBFvariousup-streamfactors_____________________________________________________TBPisauniversalfactorTranscriptionalelongationCTDphosphorylationstatusofRNApolIIStepsleadingtotranscriptionalactivationPromoterescape/clearanceTransitiontoelongationphaseWhathappensduringtranscriptionalelongation?

Originalcontactswithinpre-initiationcomplexabolished

Formationofnewcontactswithelongationfactors

PhosphorylationofCTD

ChangeofRNApolIItoaternarycomplex=highstabilityModelofnucleosomedynamicsduringtranscription

PhosphorylationoftheCTDdefinesthestageoftranscription

CTDconsistsofheptadrepeatsoftheconsensussequence:YSPTSPS

#ofrepeatsdifferinorganisms

Promoterclearance:Ser#5getsphosphorylatedCTD:Notphosphorylated

Transitiontoelongation:Ser#2getsphosphorylatedCTD:phosphorylatedExperimentalevidenceforelongationfactors

ComparisonofRNAPIIelongationrate

invitro:100-300nt/min,frequentpauses,andsometimesfullarrest

invivo:1200-2000nt/minWhythediscrepancy?

Useofpharmacologicalagents

DRB(5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole

DRB,nucleotide-analogue,causeinhibitionofhnRNAtranscriptionbyarrestingRNApolIIinvivo,butnotpurifiedRNApolII.Possibletarget?TheseevidencesuggestexistenceoffactorsthatfacilitatetranscriptionalelongationRNApolymeraseIIoftenencounterspauses&