医学分子生物学：Transcription

Transcription2016.4.15Transcription:DNA-dependentsynthesisofRNACappingandsplicing:RNAprocessingReversetranscription:retrovirusKeytopics:ReferencesMichaelM.CoxetalReferencesRobertK.MurrayBruceAlberts*Famousresearchers

inthisfieldRogerKornborg,Ph.D,Structurebiologist2006,NobelPrizeinChemistryRogerKornbergLab-StanfordUniversityOurresearchisdirectedtowardsthemechanismandregulationofRNApolymeraseIItranscription.Transcriptionisthefirststepandthekeycontrolpointinthepathwayofgeneexpression.Transcriptionalregulationunderliesdevelopment,oncogenesis,andotherfundamentalprocesses.Knownfor:Transmissionofgeneticinformationfrom

DNA

to

RNA/SelectedPublicationsNanoparticleimaging.Electronmicroscopyofgoldnanoparticlesatatomicresolution.Science,2014;345(6199):909-912ArchitectureofanRNAPolymeraseIITranscriptionPre-InitiationComplexScience,2013;342(6159):709LockandKeytoTranscription:sigma-DNAInteraction.Cell,2011;147(6):1218-1219InitiationComplexStructureandPromoterProofreading.Science,2011;333(6042):633-637StructureofanRNAPolymeraseII-TFIIBComplexandtheTranscriptionInitiationMechanism.Science,2010;327(5962):206-209StructuralBasisofTranscription:BacktrackedRNAPolymeraseIIat3.4AngstromResolution.Science,2009;324(5931):1203-1206Nucleosomeretentionandthestochasticnatureofpromoterchromatinremodelingfortranscription.Cell,2008;133(4):716-726Structureofathiolmonolayer-protectedgoldnanoparticleat1.1angstromresolutionScience,2007;318(5849):430-433Structuralbasisoftranscription:Roleofthetriggerloopinsubstratespecificityandcatalysis.Cell,2006;127(5):941-954RobertTjian,PhD（钱泽南）HHMI(TheHowardHughesMedicalInstitute)President,2009–PresentRobertTjianLab-HHMI/UCBerkeleyOurmainresearchinterestinvolvesdecipheringexactlyhowanimalcellsretrievethegeneticinformationstoredinDNAmoleculesduringtranscription–thebiochemicalprocessthatleadstotheproductionofproteins./scientists/robert-tjianSelectedPublicationsLoopingbacktoleapforward:transcriptionentersanewera.Cell.2014,157(1):13-25.Review.Single-moleculedynamicsofenhanceosomeassemblyinembryonicstemcells.Cell.2014,156(6):1274-85.ADNArepaircomplexfunctionsasanOct4/Sox2coactivatorinembryonicstemcells.Cell.2011,147(1):120-31.Controlofembryonicstemcelllineagecommitmentbycorepromoterfactor,TAF3.Cell.2011,146(5):720-31.doi:10.1016/j.cell.2011.08.005.Invitroanalysisofhuntingtin-mediatedtranscriptionalrepressionrevealsmultipletranscriptionfactortargets.Cell.2005,123(7):1241-53.Transcriptionregulationandanimaldiversity.Nature.2003,424(6945):147-51.Review.TRF2associateswithDREFanddirectspromoter-selectivegeneexpressioninDrosophila.Nature.2002,420(6914):439-45.Neurodegeneration.Aglutamine-richtrailleadstotranscriptionfactors.Science.2002,296(5576):2149-50.Structure,function,andactivator-inducedconformationsoftheCRSPcoactivator.Science.2002Feb8;295(5557):1058-62TranscriptionCentraldogmaReplicationTranscriptionTranslationReversetranscriptionRNA

replicationDNARNAProteinThepathwayfromDNAtoproteinOverviewofRNAs’FunctionRibonucleicacidsplaythreewell-understoodrolesinlivingcells:MessengerRNAsencodetheaminoacidsequencesofallthepolypeptidesfoundinthecellTransferRNAsmatchspecificaminoacidstotripletcodonsinmRNAduringproteinsynthesisRibosomalRNAsaretheconstituentsandcatalyticappropriateaminoacidsRibonucleicacidsplayseveralless-understoodfunctionsineukaryoticcells:MicroRNAappearstoregulatetheexpressionofgenes,possiblyviabindingtospecificnucleotidesequencesRibonucleicacidsactasgenomicmaterialinvirusesAndrewFireCraigMello2006NobelPrizeinPhysiology/MedicineIn1998,theAmericanscientistsAndrewFireandCraigMellopublishedtheirdiscoveryofamechanismthatcandegrademRNAfromaspecificgene.Thismechanism,RNAinterference,isactivatedwhenRNAmoleculesoccurasdouble-strandedpairsinthecell.Double-strandedRNAactivatesbiochemicalmachinerywhichdegradesthosemRNAmoleculesthatcarryageneticcodeidenticaltothatofthedouble-strandedRNA.WhensuchmRNAmoleculesdisappear,thecorrespondinggeneissilencedandnoproteinoftheencodedtypeismade.SurfingtheWebofRNA-MediatedGeneRegulationTranscriptionandReplicationTemplateOnlyonestrandTwostrandsMaterialsNTPdNTPBasepairA-U，T-A；G-CA-T；G-CPolymerase

RNA

polymeraseDNA

polymeraseProductsmRNA,tRNA,rRNA,etalDNATranscriptionReplicationFeaturesofTranscriptionRNApolymerasebindstosequencecalledpromotertobegintranscription---PrimernotrequiredDNAduplexunwinds,forminga“bubble”of~17bpRNAPolgeneratespositivesupercoilsahead,laterrelievedbytopoisomerasesThetranscription“bubble”I.TranscriptionBasicsThetranscriptionrequiresMaterials:

NTP(ATP,UTP,GTP,CTP)Template:

DNAEnzyme:RNA

polymerase(RNApolymerase,RNA-pol)OtherproteinsProducts:singlestrandRNAcontainingDNAheredityinformation5’-----GCAGTACATGTC-----3’SenseDNA3’-----CGTCATGTACAG-----5’anti-sense5’-----GCAGUACAUGUC-----3’mRNAN------Ala--Val--His--Val------CProteinOverviewofRNAs’MetabolismRibonucleicacidsaresynthesizedincellsusingDNAasatemplateintranscriptionTranscriptionistightlyregulatedinordertocontroltheconcentrationofeachproteinBeingmainlysingle-stranded,manyRNAmoleculescanfoldintocompactstructureswithspecificfunctionsSomeRNAmoleculescanactascatalysts(ribozymes),oftenusingmetalionsascofactorsMosteukaryoticribonucleicacidsareprocessedaftersynthesisEliminationofintrons;joiningofexonsPoly-adenylationofthe3’endCappingthe5’endII.RNAPolymerasesBacterialRNApolymeraseEukaryoticRNApolymeraseIIcoreenzymeholoenzyme

RNApolymeraseinbacteria

SubunitMW(kDa)Functionα37β151β’155σ70InteractswithmodulatorproteinsCatalysisBindstothetemplateRecognizesthepromoterregionω11Notfullyelucidated

ⅠⅡⅢLocationNucleolusNucleoplasmProducts45SrRNAhnRNAtRNA,5SrRNA,snRNAEukaryoticRNApolymeraseNucleoplasmSubunitsRPA1,RPA2,RPC5/RPC9,RPB6,etc.RPB1,RPB2,RPB3/RBP11,RPB6,etc.RPC1,RPC2,RPC5/RPC9,RPB6,etc.SummaryTranscriptioniscatalyedbyDNA-dependentRNApolymerases,whichuseribonucleoside5’-triphosphatestosynthesizeRNAcomplementarytothetemplatestrandofduplexDNA.ThestepsoftranscriptionconsistsofbindingofRNApolymerasetoapromoteronDNAtoformaclosedcomplex,openingofthecomplexbylocalDNAunwindingnearthepromoter.BacterialRNApolymerasesusesasigma(σ)factortorecognizeandbindthepromoterduringinitiation.EukaryoticcellshavethreetypesofRNApolymerases.PolIandPolIIItranscribegenesencodingrRNAsandsmallfunctionalRNAssuchastRNA,respectively.PolIItranscribesprotein-codinggenestomakemRNA.III.TranscriptioninBacteriaPromotersinE.ColithatbindthesameRNApolymerasehavecommonfeaturesTwoconsensussequencesat−10(TATAAT)and−35(TTGACA)forsubunitbindingCalledTATAsequencesA-T−richupstreampromoterelementbetween−40and−60bindsthesubunitThesesequencesgovernefficacyofRNAPolbindingandthereforeaffectgeneexpressionlevelFeaturesofbacterialpromotersrecognizedbyσ70

-10bp-------TATAAT(TATAbox)(pribnowbox)-35bp-------TTGACAAnoverviewoftranscriptionInitiationElongationTerminationTwoTypesofTerminationinE.Coli1)-independent-CharacterizedbythreeUsnearthe3’endofthetranscript-Self-complementaryregionsintranscriptformahairpin15-20ntbeforethe3’endMakestheRNAPolpause,dissociate2)-dependent-Known:commonCA-richsequencecalleda

rutsite(Rhoutilizationelement)-proteinprocessesuntilterminationsitereached-proteinisahelicase,bindstorutsiteSummaryTranscriptionbeginsatspecificpromoter

sequencesupstreamfromthecodingsequenceintheDNAtemplate.Duringelongation,theRNApolymeraseishighlyprocessive,synthesizingtranscriptswithoutdissociatingfromtheDNAtemplate.TerminationoccurswhenthepolymerasetranscribesthroughcertainDNAsequenceinaprocessthatsometimesrequiresanaccessoryfactor,ρ.III.TranscriptioninEukaryotesEukaryotescontainseveraldistinctpolymerasesRNApolymeraseIsynthesizespre-ribosomalRNA(precursorfor28S,18S,and5.8rRNAs)RNApolymeraseIIisresponsibleforsynthesisofmRNAVeryfast(500-1000nucleotides/sec)Specificallyinhibitedbymushroomtoxin

-amanitinCanrecognizethousandsofpromotersRNApolymeraseIIImakestRNAsandsomesmallRNAproductsPlantsappeartohaveRNApolymeraseIVthatisresponsibleforthesynthesisofsmallinterferingRNAsMitochondriahavetheirownRNApolymeraseThePolIpromoterThePolIIIpromoterFeaturesofSomePromotersRecognizedbyEukaryoticRNAPolymeraseII

-30bp-------TATAAT(TATAbox)(hognessbox)-40bp-------GCbox-110bp-------CAATboxEukaryoticmRNAtranscriptioninvolvesmanyproteinsReliesonprotein-proteincontactsManyhighlyconservedtranscriptionfactorsRNAPolIIiswell-studiedLargecomplexof12subunitsSomesubunitshavesomestructuralhomologytobacterialRNApolymeraseHascarboxy-terminaldomainofhighlyconservedrepeatsTranscriptionatPolIIpromoterTranscriptioninitiationinvivorequiresthemediatorcomplexElongationandTerminationElongationfactorsboundtoRNAPolIIenhanceprocessivityandcoordinatepost-translationalmodificationsFortermination,PolIIisdephosphorylatedRegulationiscomplexTerminationmechanismsvaryamongRNApolymerasesSummaryPolI,II,IIIrecognizedistinctpromotersequencesandrequireuniquesetsoftranscriptionfactors.Asinbacteria,transcriptioninitiationineukaryotesishighlyregulatedandincludesmultiplestepsthatleadtoassemblyofanactivepolymerasecomplexatapromoter.PolIItranscription(themoststudies)proceedsthroughdistinctphasesofassembly,initiation,elongation,andtermination.TranscriptionregulationineukaryotesisenhancedbyMediator,alargeproteincomplexthatbindssimultaneouslywithgeneraltranscriptionfactorsassociatedwithPolIIandspecifictranscriptionfactorsassociatedwithupstreamelements.山中伸弥2012年诺贝尔生理或医学奖ReferencesforExperimentsM.凯里S.T.斯梅尔著MichaelGreeenJosephSambrookIV.RNAprocessing1.ThemRNA5’CappingThe5’-capisa7-methylguanosine7-methylguanosinelinksto5’-endvia5/,5’-triphosphatelinkMayincludeadditionalmethylationsat2’OHgroupsofnexttwonucleotidesMethylgroupsderivefromS-adenosylmethionine(SAMe)ProtectsRNAfromnucleasesFormsabindingsiteforribosome2.Additionofthe3’poly(A)tailtothetranscriptPoly(A)tailisaddedto

eukaryoticmRNAstoserveasabindingsiteRNAPolIIsynthesizesRNAbeyondthecleavagesignalsequenceCleavagesignalisboundbyanendonucleaseandapolyadenylatepolymeraseboundtoCTDEndonucleasecleavesRNA10-30ntdownstreamtohighlyconservedAAUAAPolyadenylatepolymerasesynthesizes80-250ntofAInterruptedgenes3.Pre-mRNAsplicingADNA-mRNAhybridrevealingthepresentofintronsDifferentwaysofassemblingexonsAlternativeprocessingoftheratcalcitonimFourClassesofIntronsGroupIandGroupIIintronsareself-splicingRequirenoadditionalproteinsorATPInnuclear,mitochondrial,andchloroplastgenomesDiffermainlyinthesplicingmechanismSpliceosomalintronsaresplicedbyenormouscomplexescalledsplicesomesThemostcommonintronsFrequentinprotein-codingregionsofeukaryoticgenomestRNAintronsaresplicedbyprotein-basedenzymesPrimarytranscriptcleavedbyendonucleaseExonsarejoinedbyATP-dependentligaseAnoverviewofthesplicingreactionGUat5’-endandAGat3’-endusuallymarksitesofsplicingSpliceosomeintronsareremovedviaalargecomplexcalledaspliceosomeSpliceosomemadeupofsnRNPs(“snurps”forsmallnuclearribonuclearproteins)snRNPRNAiscalledsnRNA(forsmallnuclearRNA)5snRNAsknownineukaryotes(U1,U2,U4,U5,U6)BindingofU1andU2snRNPtomRNAU1helpsdefinethe5’-splicesiteU2bindsnearthe3’-endoftheintronNext,U2,U4,U5,andU6bind,bringingatleast50proteinstocreatespliceosomeATPrequiredforassemblybutnotcleavageSomepartsattachedtoCTD(carboxy-terminaldomain)ofRNAPolIIIndicatescoordinationofsplicingwithtranscriptionU1snRNPandU2snRNPbindtotheintron’sends(cont.)Coordionoftranscriptionandpre-mRNAprocessing4.RNAeditingC-to-UeditingofthemRNAforApoBAsinglegenecanyielddifferentproductsdependingonRNAprocessingRNAcanbe“edited”(basesremoved/added)Cleavage/polyadenylationpatternscanvary,yieldingdifferentmaturetranscripts5.CellularmRNAsaredegradedatdifferentratesRNAlifetimeisonemeansofgeneregulationHalf-livesvaryfromsecondstohoursTypicalvertebratemRNA~3hrs~10turnoverspercellgenerationShorter(~1.5mins)half-livesforbacterialmRNAsDegradationviaribonucleasesHairpinstructuresinmRNAcanextendhalf-lifeProcessingofnon-protein-codingRNAsNon-codingRNARNAtranscriptsProteincodingRNA(mRNA）Non-codingRNARegulatoryRNAsHousekeepingRNAsmiRNAIncRNAsiRNApiRNA

tRNAsnRNArRNAsnoRNA

ProcessingoftRNAandrRNABasesaremodifiedinpost-transcriptionrxsPseudouridine(

)ThiouridineDihydrouridine,etc.rRNAsandtRNAsarecleavedfromlongerprecursorsRNAaseP、incisionenzymeTheprocessingoftRNACleavageandsplicingtRNAnucleotidetransferase,ligaseATPADPAdditionBasemodification（2）UDHU

（3）Uψ（4）A

I（1）AAm（1）（1）（3）（2）（4）Processingofpre-rRNAtranscriptsinbacteriaMicroRNAsFunctioninGeneRegulationMicroRNAs(miRNAs):ShortnoncodingRNAsof~22nucleotidesBindtospecificregionsofmRNAtoaltertranslationAssistincleavingthemRNAsOrblockthemRNAfromtranslation~1%ofthehumangenomemayencodemiRNA!SynthesizedfromlargerprecursorsProcessedbytwoendoribonucleases,DroshaandDicerStepsinmiRNAProcessingLongprecursorpri-miRNAmadeinnucleusDroshaandDGCR8cleavepri-miRNAtoa70−80ntprecursorExportinandRanexportthisprecursortothecytoplasmDicercleavesthepre-miRNAintodsRNAComplementofmiRNAremovedbyhelicasemiRNAloadedontoproteincomplexsuchasRNA-inducedsilencingcomplex(RISC)RISCbindstotargetmRNASummaryEukaryoticmRNAsaremodifiedbytheadditionofa7-methylguanosineresidue

atthe5’endandbycleavageandpolyadenylationatthe3’endtoformapoly(A)tail.MessengerRNAcapping,polyadenylation,andsplicingarecoupledtotranscriptionthroughthephysicalassociationoftheenzymeswithRNApolymeraseII.Mosteukaryoticpre-mRNAcontainintrons,interveningsequencesthatareremovedastheflankingexonsequencesarejoinedtogetherintheprocessofsplicing.Eukaryoticpre-mRNAscancontaindozensorhundredsofintrons.Inalternativesplicing,thesplicingofdifferentsetsofintronscanproduceaseriesofdifferentmRNAsfromthesameinitialtranscript.Mostintronsaresplicedbythesplicesome,acomplexoffivesmallnuclearribonucleoproteins(snRNPs);sequenceswithinpre-mRNAmarktheexon-intronboundaries.ThenucleotidesequenceofeukaryoticmRNAsissometimesalteredbyRNAeditingenzymes.Thiscanhavefar-reachingeffects,suchasalteringtheproteinencodedbythemRNAorinfluencingtheregulationofmRNAtranslation.CellularmRNAsaredegradedatdifferentrates,dependingontheirinteractionswithribonucleases.RegulaotryRNAs,suchasmiRNAs,arealsoprocessedfromlargertranscripts.V.ReversetranscriptionReversetranscription1.Retrovirus&Reversetranscriptase(1)History1964TeminfoundRNAvirus(ASV)1970TeminandBaltimorefoundreversetranscriptase(2)ThefunctionofreversetranscriptaseReversetranscriptase

(RNAdependentDNApolymerase,

RDDP)Itsfunction:

RNAdependentDNApolymerase

RNAhydrolyse

DNA-dependentDNAsynthetaseRetrovirusescanmakeDNAfromRNARetroviruseshavegenomesofssRN