chap12Biosynthesis of RNAs 华东理工大学生物化学课件

Transcription BiosynthesisofRNAs 5theditionbyHarveyLodish MatthewP Scott PaulMatsudaira andJamesDarnell 4theditionbyHarveyLodish ArnoldBerk S LawrenceZipursky andPaulMatsudaira LehningerPrinciplesofBiochemistry FourthEdition Biochemistry5theditionJeremyM Berg LubertStryer andJohnL Tymoczko Outline Transcription thereactionTranscriptionineukaryotesTranscriptioninitiationTranscriptionfactors regulationofgeneexpressionmRNAprocessingmRNAstabilityGenepredictionTranscriptioninprokaryotesDifferencesbetweeneukaryotesandprokaryotesRNApolymeraseinE coliOperonsRegulationofgeneexpressioninprokaryotes Fertilizedegg Itwasinthispub EaglepubinCambridge JamesWatsonandFrancisCrickannouncedtheirdiscoveryofDNAonFebruary28 1953 1953 1993 Wehavediscoveredthesecretoflife Transcription Translation Centraldogma ItwasfirstproposedbyFrancisCrickin1958andre statedinaNaturepaperpublishedin1970 Transcription thereaction RNAsynthesisinvolves SeparationoftheDNAstrands UsingoneoftheDNAstrandsastemplate SynthesisofanRNAmoleculeinthe5 to3 directionbyRNApolymeraseusingA G CandU Noprimersneeded Noproofreading Incomplementarybasepairing A T G andConthetemplateDNAstrandspecifyU A C andG respectively ontheRNAstrandbeingsynthesized PolyploidchromosomesinDrosophila IfourstrandsofDNAwerestretchedoutinaline the46chromosomesmakingupthehumangenomewouldextendmorethantwometers Thehumangenomecontainsmorethan3 3billionbasepairsbutonlyabout30 000genes Question Howcancellsfindtherightsitesonthechromosomestostartranscription howlongtogoandwheretostop RNAsynthesis Likeallbiologicalpolymerizationreactions RNAsynthesistakesplaceinthreesteps initiation elongationandtermination Initiation tosearchthegenomicDNAforinitiationsites alsocalledpromotersites Forexample E coligenomicDNAhasabout4 6millionbasepairsand2000promoters Cellsmusthaveamechanismtoknowwheretostartthetranscription Althoughgeneexpressioncanberegulatedatmanysteps themostefficientcontrolofgeneexpressionisatthetranscriptioninitiationstep Elongation tounwindashortstretchofDNAtoproduceasingle strandedDNAasatemplate UsingcorrectribonucleosidetriphosphatesassubstratesandDNAastemplateRNApolymerasecatalyzestheformationofphosphodiesterbonds ThisprocessisrepeatedmanytimestoproduceanRNAmoleculeofcorrectlength Termination todetectterminationsignalsthatspecifywhereatranscriptends Transcriptioninitiationineukaryotes ReactionmixturescontainedGTP CTP ATPand H3 UTPinTris HClbuffer FourmgofnativeX laevisDNAwasusedasDNAtemplate ReactionswerestartedbyaddingproteinsolutionselutedfromaDEAE column After20minofincubationat30oCthemixturesweretransferredontoaDEAE cellulosediscsfollowedimmersionofthediscsin0 5MNa2HPO4 Thefilterswerethenwashedandtheradio activitiesofthediscsweredetermined FromJ BiolChem 249 241 1974 ThreetypesofRNApolymerasesineukaryotes TherearethreetypesofRNApolymerases Eachcontainstwolargesubunits 220kDaand 150kDaeach and10to15smallersubunits EachtypeofRNApolymerasetranscribesadifferentsetofRNAs TypeI forrRNAsynthesis mostabundantRNAs TypeII formRNAsynthesis protein codingRNAs TypeIII fortRNAand5srRNAsynthesis smallRNAs Therefore theregulationofgeneexpressionistocontrolthetranscriptioncarriedoutbytypeIIRNApolymeraseatspecificlocationsofthechromosomes ThreetypesofRNApolymerasesineukaryoticcells FactorsrequiredfortranscriptioninitiationbyRNApolymeraseII Proteinsrequired RNApolymeraseII Generaltranscriptionfactors Specifictranscriptionfactors Mediators cis DNAelements Promoters Enhancers Invitrotranscription transcriptionstartsatspecificsitesontheDNA Toplineontheleftshowstherestrictionsitesintheregionofavirusgenomewherethetranscription initiationsiteislocatedbythisnascent transcriptanalysis Differentrestrictionfragmentswereindividuallyincubatedwithanuclearextractandradio labeledribonucleosidestriphosphates TranscriptionbeginsatthestartsiteandendswhenpolIImoleculerunsoffthetemplate Thesizesoftheproductsareanalyzedbygelelectrophoresis a amanitinisaspecificpolIIinhibitor Comparisonofnucleotidesequencesupstreamoftranscriptionstartsiteinvertebrategenes Comparisonofnucleotidesequencesupstreamofthestartsitein60differentvertebrateprotein codinggenes Eachsequencewasalignedtomaximizehomologyintheregionfrom35to20 Thetabulatednumbersarethepercentagefrequencyofeachbaseateachposition Maximumhomologyoccursoverasix baseregion referredtoastheTATAbox whoseconsensussequenceisshownatthebottom TheinitialbaseinmRNAsencodedbygenescontainingaTATAboxmostfrequentlyisanA Afunctionalanalysis Theeffectofspecificpointmutationsinthemousebeta globinpromoterontheabilityofthatpromotertoinitiatetranscription Eachlinerepresentsthetranscriptionlevelofamutantpromoterrelativetothetranscriptionlevelofthewild typepromoter Startingat 106to 475 atotalof130single basemutationsweregenerated Theblackdotsindicatenomutationwasgenerated Noticethattherearethreeclustersreducedtranscriptionwhenmutated Maniatisetal Science1987 236 1237 45 Certainregionsinthepromoterisrequiredforsuccessfultranscriptioninitiation Twocommonelementsinthepromoterregion Basalelements nearthetranscriptionstart 25bp ATATbox TATAAAA AT richDNAiseasiertodenaturethanGC richDNA Proximalelements locatedupstream 50to 200bp CatBox CAATand GCBox GGGCGG Linkerscanningmutationstoidentifytranscription controlelements ThymidineKinase Promoterregionofaprotein codinggene theTATAboxandtheupstream proximal elements Atypicalpromoterregionforaprotein codingeukaryoticgene TheTATAboxisabout30bpsupstreamofthetranscriptioninitiationsite TheTATAboxbindsthegeneraltranscriptionfactorswhichdirectRNAPolymeraseIItothetranscriptioninitiationsite Severalupstreampromoterelementsmaybindothertranscriptionactivatorstofacilitatetranscription FromGilbert DevelopmentalBiology 4thedition Inthisexperiment theregionbetweentheTATAboxandtheoriginalstartpointoftranscriptionwasprogressivelyshortenedbymakingsmalldeletions Eachtimeadeletionwasmade thebeginningpointoftranscriptionwasshiftedfurtherdownstreamfromtheoriginal Thisexperimentindicatesthattranscriptionalwaysbeginsabout30basesdownstreamfromtheTATAbox Transcriptionstartsabout30basepairsdownstreamoftheTATAbox Promoterregionsofseveralgenesasexamples notallgeneshaveTATAbox Inordertobegintranscription RNApolymeraserequiresanumberofgeneraltranscriptionfactors calledTFIIA TFIIB andsoon ThepromotercontainsaDNAsequencecalledtheTATAbox whichislocated25 30nucleotidesawayfromthesitewheretranscriptionisinitiated 1 TheTATAboxisrecognizedandboundbyTATA bindingprotein TBP whichispartoftranscriptionfactorTFIID whichthenenablestheadjacentbindingofTFIIB 2 ThebindingofTFIIDcausedDNAdistortion 3someofthosefromhumancellscanbereplacedinbiochemicalexperimentsbythecorrespondingfactorsfromsimpleyeasts Transcriptioninitiation Transcriptionfactors specificgeneexpression Manygenesareexpressedinalmostallcells thesegenescanbecalled housekeepinggenes suchasgenesrelatedtoglucosemetabolism TheexpressionofthesegenesrequirespolIIandtheso calledgeneraltranscriptionfactors suchasTFIIB E F H etc Othergenes suchasinsulin hemoglobinandinterferon g areonlyexpressedinspecificcelltypes Theexpressionofthesegenesaretightlycontrolledbyspecifictranscriptionfactors Thesetranscriptionfactorsusuallycontaintwocharacteristicdomains oneDNA bindingdomainandoneactivationdomain TheDNAbindingdomainrecognizeaspecificsequenceofDNAonthechromosomeandtheactivationdomaincontrolthetranscription Morethan2000transcriptionfactorsareencodedinthehumangenome BasedonthestructuralfeaturesoftheDNAbindingdomainsofthespecifictranscriptionfactorscanbedividedintodifferentgroups Theseincludehelix turn helix zincfingerandleucinezipper etc Functionalclassificationofpositive actingtranscriptionfactors Majorfunctionalgroupsareshowninblack specificexamplesareillustratedinred Thelistofexamplesisnotexhaustive Brivanlou Darnell Science 2002 295 813 Transcriptionactivation enhancersandtranscriptionactivators EnhancersareDNAsequencesthatcangreatlyincreasethetranscriptionrate Transcriptionactivatorsareproteinsthatbindtheenhancerregionstoactivatetranscription MajorstructuralmotifsintheDNA bindingdomains helix turn helix zincfinger leucinezipper ThesetranscriptionfactorseachcontainsoneDNA bindingdomain blue andoneormoreactivationdomains green Gal4andGcn4areyeasttranscriptionfactors GR theglucocorticoidreceptor alsocontainsahormone bindingdomain SP1bindstoGC richpromoterelementsinalargenumberofmammaliangenes Lodishetal MolecularCellBiology 5thedition AtranscriptionfactorcontainsaDNA bindingdomainandaactivationdomain The3Dstructureshowsaphage434repressorproteincomplextogetherwith20basepairsofDNA notethatphage434repressorproteinisnotamammalianprotein ItisusedtoshowtheH T Hstructuralfeatures Therepressorproteincontainsthehelix turn helixmotif ItsrecognitionhelixliesinthemajorgrooveofDNA Ahomodimmerofatranscriptionactivator DNA bindingdomainandtheleucinezipperFromMcKinght SciAm1991April AzincfingerisaproteindomainthatcanbindtoDNA Azincfingerconsistsoftwoantiparallel strands andan helix Oneverywellexploredsub setofzinc fingers theC2H2class comprisesapairofcysteineresiduesinthebetasheetsandtwohistidineresiduesinthea helixwhichareresponsibleforbindingazincion InteractionbetweenaC2H2zinc fingerproteinandDNA Gal4isaC6zinc fingerprotein BindingofsixcysteineresiduestotwozincionsineachmonomerformsacompactglobulardomainthatinteractswithDNA ItbindsDNAasahomodimmer Twomonomersinteractingtoformacoiledcoilstructureholdthemonomerstogether InteractionbetweenGal4protein aC6zinc fingerprotein andDNA High leveltranscriptioncanbeachievedbybindingofatranscriptionactivatortoanenhancer 1 EnhancersequencesintheDNAcanbeasingleormultiplecopiesineitherorientation 2 Usuallylocatedupstream butitcanbedownstream orevenlocatedintheintrons 3 Canbeseveralkilobasesawayfromthepromoter 4 Silencerelementsandrepressorfactorsalsoexisttosuppressgeneexpression AnexpressionconstructtospecificallyexpressthegeneofinterestinthedevelopingeyeinDrosophila A anormalDrosophilaeye B Acelldeath inducinggeneisexpressedunderthecontrolofaneye specificpromoter Theflyisnormalexceptthatitdoesnothavetheeyes C boththecelldeathgeneandadeathinhibitorgeneareexpressedunderthesamepromoterintheeye D thecelldeathinhibitoraloneisexpressedintheeye Expressionofacelldeathgeneundertheeyespecificpromoter A C D B Somemajorsignalingpathwaysthatdeliveranactivetranscriptionfactortothenucleusinresponsetocellsurfacereceptorbindingbyanextracellularproteinligand Thesefactorsareactivatedbypolypeptideligand receptorinteractionatthecellsurfaceandthenaccumulateinthenucleustoregulatethetranscriptionoftargetgenes Transcriptionelongationandtermination Elongation thecarboxylterminaldomain CTD ofthelargestsubunitofRNAPolIIcontains 50repeatsofaseven amino acidpeptide Tyr Ser Pro Thr Ser Pro Ser TSPTSPS Astranscriptionstartsandtranscribeawayfromthepromoter theCTDisphosphorylatedandthegeneraltranscriptionfactorsdissociatefromtheinitiationcomplex RNAPolIIthensynthesizestheprimaryRNAasitmovesalongtheDNAtemplate Termination RNAPolIIterminatesatmultiplesitesinthelastexonbeyondthepoly A signal Itcanbe0 5to2kbdownstreamfromthepoly A additionsite Theterminationmaybecoupledtothecleavageandpolyadenylationprocess TheC TerminalDomain CTD ofRNAPolIIconsistsof52repeatsoftheconsensusheptapeptideTSPTSPSandservesasaplatformfortheorderedassemblyofthefactorsresponsiblefortranscription pre mRNA5 capping splicing and3 processingatdifferentstagesinthesynthesisofthenascenttranscript Orphanides ReinbergCell2002 108 439 451 TheC TerminalDomain CTD ofRNAPolymeraseIICoordinatesTranscriptionandPre mRNAProcessing mRNAprocessing Thecap whentheRNApolymerasehastranscribedonlyabout20 30nucleotides thefree5 endofthetranscriptiscappedwith7 methylguanosinethrougha5 5 triphosphatelinkage Itisessentialfortheribosometobindtothe5 endofthemRNA Poly A tail transcriptionbyRNApolymeraseIIterminatesatoneofthepossibleterminationsitesdownstreamfromthepoly A siteinthefinalexon ThentheprimaryRNAiscleavedatthepoly A additionsitewhichis 20nucleotidesdownstreamofthepolyadenylationsignal AAUAAA 100 250Aresiduesareaddedtothe3 end Splicing theintronsaresplicedoutafterthetranscriptionhascompletedforshortmRNAs Forlongeronessplicingmayoccurastranscriptionisstillgoing Thesequencesaroundthe5 and3 endoftheintronsarenearly100 conservedamongvertebrategenes TheintronsalwaysbeginwithGUat5 endandendwithAGatthe3 end AnAresiduelocated20 50bfrom3 endintheintronisessentialforsplicingreactiontooccur ProcessingofmRNA OverviewofRNAprocessingineukaryotesusingb globingeneasanexample Structureofthe5 methylatedcapofeukaryoticmRNA Thedistinguishingchemicalfeaturesarethe5 5linkageof7 methylguanylatetotheinitialnucleotideofthemRNAmoleculeandthemethylgrouponthe2 hydroxyloftheriboseofthefirstnucleotide base1 Boththesefeaturesoccurinallanimalcellsandincellsofhigherplants yeastslackthemethylgrouponbase1 Theriboseofthesecondnucleotide base2 alsoismethylatedinvertebrates Polyadenylation Schematicrepresentationofpoly A signalsinanimals AAUAAA poly A signal Poly A site cleavagesite DSE U richorGU richdownstreamelements nt nucleotides Zhaoetal MicrobiolMolBiolRev1999Jun 63 2 405 45 Polyadenylationatthe3 endofmRNAs Cleavageandpolyadenylationofpre mRNAsinmammaliancells Cleavageandpolyadenylationspecificityfactor CPSF bindstotheupstreamAAUAAApoly A signal CStFinteractswithadownstreamGU orU richsequenceandwithboundCPSF formingaloopintheRNA BindingofCFIandCFIIhelptostabilizethecomplex Poly A polymerase PAP cleavestheRNAatthepoly A sitewhichis10 30bpsdownstreamofthepoly A signalandtheinitial 12Aresiduesareadded Bindingofpoly A bindingproteinII PABPII totheinitialshortpoly A tailacceleratetherateofadditionofAsbyPAP 3 endUTRsofhumanIgG1cDNAstoshowthatpolyadenylationstarts 20bpsdownstreamoftheAATAAAsite Above consensuspolyadenylationsignalBelow polyadenylationefficiencyofeachvariants Efficiencyofdifferentsequencevariantsaspolyadenylationsignals TIBS 1990 July Somegeneshavemorethanonepolyadenylationsites GenomeResearch 2000 10 1001 1010 Comparingmorethan4000ESTsequenceswithgenomicsequencesrevealsthatmorethan20 ofthegenescontainmorethanonepoly A sites mRNAsplicing Intronsandexons Eukaryotepre mRNAsoftenhaveinterveningintronsthatmustberemovedduringRNAprocessing intron non codingDNAsequencesbetweenexonsinagene exon expressedDNAsequencesinagene codeforaminoacids 1993 RichardRoberts NewEnglandBiolabs PhillipSharp MIT ElectronmicroscopyofanRNA DNAhybridbetweenadenovirusDNAandthemRNAencodingthehexonprotein ModifiedfromPNAS1977 74 3171 mRNAsplicingofexonsandremovalofintronsIntronstypicallybeginwitha5 GT U andendwithAG 3 Cleavageoccursfirstatthe5 endofintron1 between2exons ThenowfreeGjoinswithanAataspecificbranchpointsequenceinthemiddleoftheintron usinga2 to5 phosphodiesterbond Intronformsalariat shapedstructure Lariatisexcised andtheexonsarejoinedtoformasplicedmRNA Splicingismediatedbysplicosomes complexesofsmallnuclearRNAs snRNAs andproteins thatcleavetheintronatthe3 endandjointheexons Intronsaredegradedbythecell Inthefirstreaction theesterbondbetweenthe5 phosphorusoftheintronandthe3 oxygen red ofexon1isexchangedforanesterbondwiththe2 oxygen darkblue ofthebranch siteAresidue Inthesecondreaction theesterbondbetweenthe5 phosphorusofexon2andthe3 oxygen orange oftheintronisexchangedforanesterbondwiththe3 oxygenofexon1 releasingtheintronasalariatstructureandjoiningthetwoexons Arrowsshowwheretheactivatedhydroxyloxygensreactwithphosphorusatoms Splicingofexonsinpre mRNAoccursviatwotransesterificationreactions SimplifiedmRNAsplicingreaction ComparisonbetweenthegenomicDNAandthecDNAforChinesehamsterEF1a SummaryofRNAprocessingineukaryotesusingb globingeneasanexample mRNAstability AftertheprimaryRNAareprocessedtomaturemRNA theyaretransportedfromthenucleustothecytoplasmfortranslation Theyareeventuallydegradedinthecytoplasm ThehalflifeofdifferentmRNAvarieswidely fromminutestodaysevenmonths Alteringthestabilitymayalsobeimportantforcontrollingeukaryoticgeneexpression Thecap them7pppGaddedtothe5 endofthemRNAmayhavearoletoprotecttheRNAfrombeingdegraded Thepoly A tail thepoly A tailhasaprotectionroleonmRNA Ifthepoly A signal AAUAAA ismutatedandthepolyadenylationwillnotoccurthetranscriptswillbedegradedrapidly 3 Un TranslatedRegions UTRs somemRNAsneedtobedegradedinordertorapidlydownregulatethecorrespondingproteins The3 UTRsofthesemRNAscontainsequencemotifsAUUUAwhichmayplayaroleinRNAdegradation Theseproteinsincludecytokines lymphokinesandsometranscriptionfactors The3 UTRsofmanyunstablemRNAscontainAUUUAmotifs Sequencesof3 UTRsofsomemRNAsencodinglymphokineswhichcoordinatecell cellinteractionsduringimmuneresponse Whentheseproteinsarenolongerneeded thetranscriptionwillbeshutdown ThemRNAsalsohaveashortlifetime The3 UTRsofmostlymphokinesandsomecellcyclerelatedtranscriptionfactorscontainaconservedAUUUAsequence TheyappeartoplayaroleinregulatingthestabilitiesofmRNAs GMCSF UAAUAUUUAUAUAUUUAUAUUUUUAAAAUAUUUAUUUAUUUAUUUAUUUAAIFN beta UUGAAAUUUUUAUUAAAUUAUGAGUUAUUUUUAUUUAUUUAAAUUUUAUUUIFN gamma UAAAUCUAUUUAUUAAUAUUUAACAUUAUUUAUAUIL 1 UUAUUUUUUAAUUAUUAUUUAUAUAUGUAUUUAUAAAUAUAUUUAAGAUAAIL 2 UUCUAUUUAUUUAAAUAUUUAAAUUUUAUAUUUAUUGUUTNF AUUAUUUAUUAUUUAUUUAUUAUUUAUUUAUUUAIFNA2 AUUAUCUAUUUAAAUAUUUUUAAAAUAUUAUUUAUUUAACUAUUUAUAAAAIFNA4 UCUAUUUAUUUAAAUAUUUAUUUAUUUUUAAAAUUUAAAUUAUUUUU DestabilizingeffectofAUUUAsequencesinthe3 UTRonmRNAhalflife Cellsweretransfectedwithdifferentexpressionvectorsandthehalflife t1 2 wasdetermined Thehalflifeofbeta globinmRNAwasabout10hours Insertionofapotionofthe3 UTRfromGMCSFwhichcontainsAUUUAreducedthehalflifeto1 2hours InsertionofthesameGMCSFregionhoweverwiththeA UresiduesreplacedwithG Cresiduesdidnotaffectthehalflifeofbeta globinmRNA Shaw Kamen Cell 1986 46 659 67 CCCTGACAAGCTGCCAGGCAGGTTCTCTTCCTCTCACATACTGACCCACGGCTCCACCCTCTCTCCCCTGGAAAGGACACCATGAGCACTGAAAGCATGATCCGGGACGTGGAGCTGGCCGAGGAGGCGCTCCCCAAGAAGACAGGGGGGCCCCAGGGCTCCAGGCGGTGCTTGTTCCTCAGCCTCTTCTCCTTCCTGATCGTGGCAGGCGCCACCACGCTCTTCTGCCTGCTGCACTTTGGAGTGATCGGCCCCCAGAGGGAAGAGTTCCCCAGGGACCTCTCTCTAATCAGCCCTCTGGCCCAGGCAGTCAGATCATCTTCTCGAACCCCGAGTGACAAGCCTGTAGCCCATGTTGTAGCAAACCCTCAAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTCCTGGCCAATGGCGTGGAGCTGAGAGATAACCAGCTGGTGGTGCCATCAGAGG