复旦大学生物化学课件生物化学RNA的生物合成和加工

Chapter16TranscriptionandPosttranscriptionalProcessingThetermtranscriptioninmolecularbiologymeansRNAbiosynthesis.Chapter161In1955FrancisCrickhypothesizedthattherewere

intermediatemoleculeswhichparticipatedintrans-ferringgeneticinformationfromDNAtoprotein.TheintermediatemoleculewasRNA.In1958heputforththefamouscentraldogma.-----DNA--------RNA------protein--------Temin1970In1955FrancisCrickhypothes2In1961theDNAdependentRNApolymerasewasdiscoveredinEcoliandthestudyonthemechanismoftranscription

began.In1982ThomasCechdiscoveredthatoneoftheprecursorRNAinTetrahymenachouldactasacatalystandcatalyzedself-splicing.Itisaribozyme.In1961theDNAdependentRNA3Section1DNAdependentRNAsynthesisRNAsynthesisiscatalyzedbyDDRP(orsimplyRNApolymerase.)

4CharacteristicsofRNAsynthesisTemplateDNA(onestrandDNAindsDNA,templatestrand)SubstratefourkindsofNTPEnzymeDDRP(noreqirementofprimer,noproof-readingfunction)DirectionofRNAchain5’to3’synthesisBasepairingGCTAAUrule

InorganicMg++Zn++ionCharacteristics5Theprocessoftranscriptioncanbedividedintothreestages:initiation,elongationandtermination.seethefig.ontheblackboard复旦大学生物化学课件生物化学RNA的生物合成和加工6ThetwostrandsindsDNAarecomplementary.Theyarethecodingstrandandthetemplatestrand.ThetemplatestrandworksasthetemplateforthesynthesisofRNA.ThesynthesizedRNAiscomplementarytothetemplatestrand.ItssequenceisthesameasthatofthecodingstrandwiththeexceptionofthesubstitutionofUforT.ThetwostrandsindsDNAarec7eg.5’CGCATTAACG3’codingstrand3’GCGTAATTGC5’templatestrand5’CGCAUUAACG3’RNAtranscriptTheReaction(NMP)n+NTP----------(NMP)n+1+PPieg.5’CGCATTAACG3’8AsymmetricTranscription-------------------------------________________________________________________________________________________________________

---------------------IndsDNAonestrandiscodingstrand.Itisnottranscribed.Theotherstrandistemplatestrand.Itistranscribed.InasegmentofdsDNAthecodinginformationofgenesmaybeindifferentstrands.

9RNApolymeraseThereisonlyonetypeofRNApolinprokaryoteswhiletherearethreetypesofRNApolsineukaryotes.EcoliRNApolymeraseEcoliRNApolcatalyzessynthesisofalltheRNAs(mRNA,rRNA,tRNAetc)

10StructureofEcoliRNApolymeraseTheholoenzymeofEcoliRNApolcontainsα2ββ’(ω)σsubunitsTheσsubunitcanbedissociatedfromtheholoenzyme.TheRNApolwithoutσsubunitiscalledcoreenzyme:α2ββ’(ω)StructureofEcol11FunctionoftheSubunit

σcanrecognizepromotersandinitiatetranscription.αcanbindregulatoryproteinsandcontrolthetranscriptional.rate.β+β’catalyzesRNAsynthesis.Theremaybeωsubunit.Itsfunctionisunknown.Therearedifferentkindsofσsubunits.Themostcommononeisσ70.Function12EukaryoticRNApolymerasesRNApolIRNApolIIRNApolIII____________________________________________________Product45SrRNAhnRNAtRNAsynthesized(precursor(precursor5SrRNAprecursorsof18S,28SofmRNA)snRNA5.8SrRNAs)____________________________________________________Sensitivitytonohighintermediate

α-amanite____________________________________________________

13StructureofEukaryoticRNApolsEachtypeofeukaryoticRNApolscontainstwodifferentlargesubunitsandtenoddsmallsubunits.ThelargestsubunitofRNApolIIcontainsconsensussequenceatthecarboxylterminalcalledCTD.CTDiscomposedofseveraldozensofheptad(YSPTSPS)repeats.Struc14CTDFunctionRNApolIIwiththeunphosphorylatedCTDparticipatesinthebeginningoftranscriptionalinitiation.DuringtheprocessofintiationmanySerandsomeTyrresiduesofCTDarephosphorylated.RNApolIIwiththephosphorylatedCTDfulfillstheinitia-tionandleavesthepromoter.RNAsynthesisentersinthestageofelongation.

15ProkaryoticTranscriptionalInitiationProkaryoticRNApolbindsthepromoterandinitiatestranscrip-tion.Promoter:DNAsequencethatisusuallyupstreamofagene’scodingsequenceandthatRNApolbindsandinitiatestranscrip-tion.σsubunitcanrecognizethepromoterEcolipromotersextendfrom–70to+30oftheinitiationsite(+1)Prokary16Mostofthemhavetworegionsofconsensussequence,the-35region(TTGACA)andthe-10region(PribnowboxTATAAT).σ70canrecognizetheconsensussequence.Somepromotersofgeneswithhightranscriptionalratehaveanotherconsensusregion,theAT-richup-element(-40to-60)whichαsubunitbinds.TheholoenzymeofRNApolbindsthepromoterviaσsubunit.Mostofthemhavetworegions17strongpromoter/weakpromoterItisthe-35and-10regionsandthedistancebetweenthemandthedistancebetween–10regionandthetranscriptionnalinitiationsite,thatdeterminesthetranscriptionalrate.Themoresimilarthe–35and–10regionsofapromotertotheconsensussequencesofTTGACAandTATAAT,thestrongeraffinityithasforRNApolbinding.Thatresultsinthehighertranscriptionalrate.Andviceversa.strong18ProcessofProkaryoticTrancriptionalInitiationRNApolrecognizesandbindsthepromoter.Thatformsaclosetranscriptioncomplex.DNAdoublehelixnear–10regionunwinds.Thatresultsinanopentranscriptioncomplex.Transcriptionbegins.Atriple-elementcomplexofDNA,RNApolandthenewlysynthesizedRNAformsTheformationofthetriple-elementcomplexcausescon-formationchange.ProcessofProkaryo19RNApolleavesthepromoterandelongationbegins.AstheelongationbeginstheσsubunitdissociatesfromRNApol.ItisthecoreenzymeofRNApolthatisresponsiblefortheelongation.

RNApolleavesthepromoteran20EukaryoticTranscriptionalInitiationEukaryoticRNApolsalonecannotinitiatetranscription.Onlywiththehelpoftranscriptionfactorscantheyfulfillthetaskofinitiation.TranscriptionalinitiationofRNApolIIneedsnotonlytheenzymebutalsomultipletranscriptionfactors.EukaryoticT21ThereareconsensussequencesinmanyoftheRNApolIIrecognizedpromoters.Theyare–30region(TATAbox)and+1region(theini-tiationsite,initiator,Inr).TheinitiationstageofRNApolIIcanbedividedintotwosteps:theassemblystepandtheinitiationstep.Thereareconsensussequences22TheAssemblyStepTBP(TATA-bindingprotein)bindsTATAbox.TFIIB(orwithTFIIA)bindsTBPandpromoter.WiththehelpofTFIIF,RNApolII-TFIIFcomplexinter-acteswithTFIIBandbindsTFIIBandpromoter.ThenTFIIEandTFIIHjointhem.RNApolIIandtheTFIIfactorsformacloseinitiationcom-plexonthepromoter.

23TheInitiationStepTFIIHhasboththehelicaseandthekinaseactivities.ItunwindsdsDNA.Thecloseinitiationcomplexbecomestheopeninitiationcom-plex.TFIIHalsocatalyzesphosphorylationofCTD.RNApolIIwithphosphorylatedCTDinitiatestranscription.

24TheInitiationStageofRNApolIorRNApolIIITheinitiationstageofRNApolIorRNApolIIIissimilartothatofRNApolII.Thetranscriptionfactorsrecognizeandbindthepromoter.RNApolIorRNApolIIIjoinsthemtoformaninitiationcomplex.Initiationbegins.TheInitiationStage25InitiationStageofRNApolIrDNA(containsgenesof18SrRNA,28SrRNAand5.8SrRNA)istranscribedbyRNApolI.Theproductis45SrRNATherearetwoconsensussequencesinrDNApromoter:coreelement(+1region)andUCE(upstreamcontrolelement).TranscriptionfactorUBFbindsUCEfirst.ThentranscriptionfactorSL1bindscoreelement.RNApolIjoinsthem.Theyformacomplexonpromoter.Initiationbegins.Initi26RNApolIIIinitiatestranscriptionof5SrRNAgeneortRNAgene.Thereareinternalpromotersinsuchgenes.Internalpromotermeansthepromoterwithinthecodingregionofthegene.tRNAgene’spromoterhastwoconsensussequences:AboxandBbox,while5SrRNAgene’spromoterhasonlyonecon-sensussequence:Cbox.RNApolIIIinitiatestranscri27TranscriptionalInitiationof5SrRNAGeneTFIIIAbindsCbox.TFIIICandTFIIIBbindTFIIIA.RNApolIIIbindsthem.Acomplexisformed.RNApolIIIinitiatestranscription.Transcriptional28InitiationRNApolIRNApolIIIRNApolII__________________________________________________ATPrequirementnonoyes__________________________________________________AandBorTATAboxcoreconsensussq.coreelementCboxInr__________________________________________________CAATboxupstreamelementUCEGCboxetc__________________________________________________generalTFsSL1TFIIIABCvariousTFIIs___________________________________________________upstreamfactorsUBFvariousup-streamfactors_____________________________________________________InitiationRNAp29TranscriptionalTerminationTherearetwotypesoftranscriptionalterminationinprokaryotes

ρindependentandρdependent.ρindependentterminationTherearetwocharacteristicsofρindependentterminationsq.(terminator).AsegmentofGC-rich,self-complementarysq.ItisfollowedbyaseriesofT.egGCCGCCAGTTCGGCTTGCCGCCTTTTTr30TheRNAsynthesizedisalsoself-complementaryandformsastem-loopstructurefollowedbyaseriesofU.

UU5’GCCGCCAGCCGGCGGUCUUGGUUU3’RNApolinteractswiththestructureandstopsatthetemplateTheUApairsareunstable.ThenewlysynthesizedRNAre-leasesfromthetemplate.Transcriptionterminates.TheRNAsynthesizedisalsose31

ρdependentterminationTheterminatoroftheρdependentterminationisnottypical.ItcontainsCA-richregionwhichρfactorcanrecognize.ρfactorisahomo-hexamerproteinfactor,canbindthenewlysynthesizedRNAandmovestotheRNA-DNAhybridregion.ρfactorhashelicaseactivityandunwindstheRNA-DNAhelixdependingontheenergyreleasedfromATPhydrolysis.ρ32Section2PosttranscriptionalProcessingMatureeukaryoticmRNAhasexperienced5’-and3’endprocessingandsplicing.hnRNA(ormRNA)iscappedat5’endMosthnRNAhaveacapof7-methylguanosinetriphosphate(m7Gppp-)atthe5’end.Itisaddedtothe5’endofthegrowingtranscriptof25-30nucleotidesvia5’5’triphosphatelinkage.ThecapprotectshnRNA(andmRNA)fromRnaseattackandparticipatesinthebindingofmRNAandribosome.

Section2Pos33hnRNA(ormRNA)hasapolyAtailatthe3’endAlmostallofeukaryotichnRNAs(ormRNAs)haveapolyAtailof80-250nucleotidesatthe3’end.Itisenzymaticallyaddedtotheprimarytranscriptintwostages.CleavageThetranscriptiscleaved10to30nucleotidespastahighlyconservedAAUAAAsq(thepolyadenylationsignalsq)andwithin50nucleotidesbeforeaGU-richsq.hnRNA(ormRNA)3410-30nts<50nts-----------AAUAAA----------------I-------------------GU-rich—cleavagesitePolyadenylationThepolyAtailissubsequentlygeneratedfromATPthroughstepwiseactionofpolyApolymerase.Multipleenzymesandproteinfactorsparticipateinthetwostages.

35Splicing.SplitGene,ExonandIntronEukaryoticgenesconsistofalternatingcodingandnon-codingsequences.Inotherwordsthecodingsqofeu-karyoticgenesisnotcontinuous.Theyaresplitgenes.Thecodingsqinthesplitgeneiscalledtheexon,thenon-codingsq,theintron.

36hnRNAisco-lineartoitstemplateDNAexceptthecapandthetail.Italsocontainsalternatingexonsandintrons.ThecodingsqofmRNAiscontinuous.hnRNAfollowingexcisionofintronsandconnectionofexonsbecomesmRNA.Thisprocessiscalledsplicing.hnRNAisco-lineartoitstem37TheMechanismofSplicingThereareconsensussequencesatthejunctionsofexonandintron.Theyarecalledsplicesites.Theshortestsplicesitesatthe5’end(splicedonor)and3’end(spliceacceptor)oftheintronareGUandAGrespec-tively.UpstreamofAGthereisabranchpointA.exon1intronexon2========GU-----------------------A-----AG=============splicedonorbranchpointspliceacceptorThe38ThebranchpointAinitiatesnucleophilicattackatthe5’endoftheintron.Itisthefirsttransesterificationreaction.

------------------=========GU-----------------A-------AG=============Exon1isreleased.Intronformsalariat.-----G==========OH-----A------AG=============ThebranchpointAinitiatesn39The3’endofexon1initiatesnucleophilicattackatthe3’endoftheintron.Itisthesecondtransesterificationreaction.===========OH>

G-----A------AG=============Exon1andexon2areconnected.Lariatformofintronisre-leased.Splicingiscompleted.=============================G-----A------AGThe3’endofexon1initiates40Thepreviouslydescribedsplicingtakesplaceinthespliceosome.SpliceosomecontainssnRNPswhicharecomposedofsnRNAsandproteins.snRNAsinsnRNPsareU-rich,andcalledURNAs.Thereare5URNAs..Theprocessofsplicinginthespliceosomeincludesspliceo-someassembly,spliceosomeactivationandsplicingThepreviouslydescribedsplic41AneukaryoticgenemaydirecttranscriptionofdifferenthnRNAsfromdifferentpromotersorusingdifferentpoly-adenylationsites.Therearetwopromotersinglucokinasegene.Inthelivercelltranscriptioninitiatesfromthesecondpro-moterwhichisnearthecodingsq,whileinthepancreaticβcelltranscriptioninitiatesfromthefirstpromoterwhichisupstreamlocated.ThisisanexamplethatonegenecanexpressdifferenthnRNAs(andmRNAs).

Aneukaryoticgenemaydirect42InthedifferentdevelopmentalstagesoflymphocyteB3’endprocessingofthehnRNAofμchaincanusedif-ferentpolyadenylationsitesandyielddifferenthnRNAS(andmRNA)Thisisanotherexample.AlternativeSplicingProvidesforDifferentmRNAsfromtheSamehnRNAThemechanismofalternativesplicingincludestheselectiveinclusionorexclusionofexons,theuseofalternative5’do-noror3’acceptorsites.Inthedifferentdevelopmental43exon1exon2exon3========-----------=====-------=============hnRNAsplicing1(mRNA1)splicing2(mRNA2)exon1exon2exon2exon3===============================splicing3(mRNA3)exson1exon2exon3===========================exon1e44hnRNA===========-------======------------===============2donorsitesinexon12acceptorsitesinexon33mRNAs===============================exon1+2+3==========================partofexon1+e2+e3=======================e1+e2+partofexon3

45rRNAprecursorprocessingrequiresRnasesprokaryotic30SrRNAprecursor----methylation----Rnasescut----precursorsof23SrRNA,16SrRNA5SrRNAandtRNA----Rnasescut----23SrRNA16SrRNA,5SrRNAandtRNArRNAprecursorprocessin46eukaryotic45SrRNAprecursor----methylation-----snoRNPmediatedprocessing----precursorsof18SrRNA,28SrRNA,5.8SrRNA----snoRNPmediatedprocessing----18SrRNA,28SrRNA,5.8SrRNAeukaryotic45SrRNAprecur47tRNAprecursorprocessingtRNAprecursorprocessinginprokaryotesandeukaryotesaresimilar.Itincludes5’endcutting3’endcuttingandCCAadding(ifthereisn’tCCA)splicing(ifthereisintron,splicingtakesplaceviaenzymaticcuttingandjoining)basemodification.

tRN48Self-splicingCatalyzedbyRNAIn1982T.CechdiscoveredthattheintronofTetrahymenarRNAprecursorcouldcatalyzedself-splicing.Theintronthatcancatalyzeself-splicingbelongstothegroupIintron.GroupIintroncatalyzesself-splicingwiththehelpofcofactorguanosineorguanosinephosphateSelf-s49Theself-splicingincludestwotransesterficationreactions5’=============---------------------==============3’exon1exon25’=============----------------------==============3’G-OHG-OHattacksthefirstnucleotideatthe5’endoftheintron.Exon1isreleased.5’=============OHG--------------------============3’Theself-splicingincludestwo50Exon1’s3’endattacksthenucleotideatthe3’endoftheintron.G----------------=================3’5’==============OHTwoexonsareconnected.Theintronisreleased.5’================================3’G---------------OHExon1’s3’endattacksthenuc51TherearegroupIIintrons,whichcanalsocatalyzeself-splicing.Theycatalyzeself-splicingjustlikethepreviouslymentionedlariat-formsplicing.Italsoincludestwotransesterificationreactionsbutdoesnottakeplaceinspliceosomeanddoesnotrequireanycofactor.GroupIandgroupIIintronsareribozymes.TherearegroupIIintrons,w52SomemRNAsUndergoEditingCodinginformationcanbechangedatthemRNAlevelbyRNAediting.InsuchcasesthecodingsqofthemRNAdiffersfromthatinthecognatDNA.RNAeditingisdiscoveredfirstinprotozoanandmediatedbyg-RNA.SomemRNAs53SomenucleotidescanbechangedinoraddedintoordeletedfromthemRNAcodingsqbyRNAediting.Thatresultsinchangeofthegeneticcodonand/orthereadingframeofthecodingsq.Theexpressedproteinischanged.Somenucleotidescanbechange54AnexampleofhumanRNAeditingistheapolipoproteinBmRNA.InliverthesingleapoBgeneistranscribedintoanmRNAthatdirectsthesynthesisofa100Kdaprotein.IntheintestinethesamegenedirectsthesynthesisoftheprimarytranscripthoweveracytidinedeaminationconvertsaCAAcodoninthemRNAtoUAAatasinglespecificsite.Ratherthanencodingglutaminethiscodonbecomesstopcodonanda48Kdaproteinistheresult.AnexampleofhumanRNAeditin55Section3RNAdependentRNAsynthesisRNAasgeneticmaterialAllplantviruses,severalbacteriophagesandmanyanimalviruseshavegenomesconsistingofRNA.TherearethreetypesofRNAgenomes:dsRNA,ssRNAandtwocopiesofthesamessRNA.ThedsRNAandssRNAarereplicatedbyRNAreplicase(RNAdependentRNApolymerase,RDRP).Section3RNAde56InmostcasesRNAgenomeissinglestranded.TherearetwosubtypesofssRNAgenomesthe+ssRNAandthe–ssRNA.+ssRNAgenomefunctionsbothasgeneticmaterialandmRNA,while–ssRNAgenomeservesonlyasgeneticmaterial.ViruseswithssRNAgenomesusethessRNAasatemplateforthesynthesisofacomplementstrand,whichcanthenserveastemplateinreplicatingtheoriginalstrand.InmostcasesRNAgenomeiss57RetroviruseshavetwocopiesofthesamessRNAasthegenome.Theycarrythereversetranscriptase.Ithasthreeenzymaticactivities:RDDP,DDDPandRnaseHRetroviruseshavetwocopieso58Chapter16TranscriptionandPosttranscriptionalProcessingThetermtranscriptioninmolecularbiologymeansRNAbiosynthesis.Chapter1659In1955FrancisCrickhypothesizedthattherewere

intermediatemoleculeswhichparticipatedintrans-ferringgeneticinformationfromDNAtoprotein.TheintermediatemoleculewasRNA.In1958heputforththefamouscentraldogma.-----DNA--------RNA------protein--------Temin1970In1955FrancisCrickhypothes60In1961theDNAdependentRNApolymerasewasdiscoveredinEcoliandthestudyonthemechanismoftranscription

began.In1982ThomasCechdiscoveredthatoneoftheprecursorRNAinTetrahymenachouldactasacatalystandcatalyzedself-splicing.Itisaribozyme.In1961theDNAdependentRNA61Section1DNAdependentRNAsynthesisRNAsynthesisiscatalyzedbyDDRP(orsimplyRNApolymerase.)

62CharacteristicsofRNAsynthesisTemplateDNA(onestrandDNAindsDNA,templatestrand)SubstratefourkindsofNTPEnzymeDDRP(noreqirementofprimer,noproof-readingfunction)DirectionofRNAchain5’to3’synthesisBasepairingGCTAAUrule

InorganicMg++Zn++ionCharacteristics63Theprocessoftranscriptioncanbedividedintothreestages:initiation,elongationandtermination.seethefig.ontheblackboard复旦大学生物化学课件生物化学RNA的生物合成和加工64ThetwostrandsindsDNAarecomplementary.Theyarethecodingstrandandthetemplatestrand.ThetemplatestrandworksasthetemplateforthesynthesisofRNA.ThesynthesizedRNAiscomplementarytothetemplatestrand.ItssequenceisthesameasthatofthecodingstrandwiththeexceptionofthesubstitutionofUforT.ThetwostrandsindsDNAarec65eg.5’CGCATTAACG3’codingstrand3’GCGTAATTGC5’templatestrand5’CGCAUUAACG3’RNAtranscriptTheReaction(NMP)n+NTP----------(NMP)n+1+PPieg.5’CGCATTAACG3’66AsymmetricTranscription-------------------------------________________________________________________________________________________________________

---------------------IndsDNAonestrandiscodingstrand.Itisnottranscribed.Theotherstrandistemplatestrand.Itistranscribed.InasegmentofdsDNAthecodinginformationofgenesmaybeindifferentstrands.

67RNApolymeraseThereisonlyonetypeofRNApolinprokaryoteswhiletherearethreetypesofRNApolsineukaryotes.EcoliRNApolymeraseEcoliRNApolcatalyzessynthesisofalltheRNAs(mRNA,rRNA,tRNAetc)

68StructureofEcoliRNApolymeraseTheholoenzymeofEcoliRNApolcontainsα2ββ’(ω)σsubunitsTheσsubunitcanbedissociatedfromtheholoenzyme.TheRNApolwithoutσsubunitiscalledcoreenzyme:α2ββ’(ω)StructureofEcol69FunctionoftheSubunit

σcanrecognizepromotersandinitiatetranscription.αcanbindregulatoryproteinsandcontrolthetranscriptional.rate.β+β’catalyzesRNAsynthesis.Theremaybeωsubunit.Itsfunctionisunknown.Therearedifferentkindsofσsubunits.Themostcommononeisσ70.Function70EukaryoticRNApolymerasesRNApolIRNApolIIRNApolIII____________________________________________________Product45SrRNAhnRNAtRNAsynthesized(precursor(precursor5SrRNAprecursorsof18S,28SofmRNA)snRNA