哈佛分子生物学讲义-蛋白质翻译(第一讲)

TranslationIOverviewLecture1Eventsoftranslation:initiation,elongation,termination,foldingMachineryinvolvedintranslationtRNAs,synthetases,ribosomes,translationfactorsGeneralregulatorymechanismsLecture2InitiationLecture3Elongation,termination,proteinfoldingregulationMethodsforstudyingtranslationTranslation-bigpictureInitiation:RecruitfMet-tRNAMet,mRNA,largeparticleElongation:SynthesizeproteinTermination:Stopsynthesis,releaseproteinEventsofTranslationInitiation(verydifferentbetweenprokaryotesandeukaryotes)DissociationofribosomeBindingofinitiationfactor(IF1/eIF1A)toA-siteofsmallsubunitBindingofotherfactorsRecruitmentoff-Met-tRNAMet

toP-siteofsmallparticleRecruitmentofmRNAtosmallparticleBindingoflargeparticle-dissociationofinitiationfactorsElongation(similarbetweenpro-andeukaryotes)Entryofaa-tRNAtoA-sitePeptide-bondformationTranslocationofmRNAandtRNAstoPandEsitesEntryofnextaa-tRNAtoA-siteetc.Termination(similarbetweenpro-andeukaryotes)EntryofreleasefactortorecognizeterminationcodonExitofpolypeptideandreleasefactorsProteinfoldingPlayersoftranslationRibosome(RNA,proteins)mRNAtRNAAminoacyl-tRNAsynthetasesTranslationfactors(initiation,elongation,termination)DifferencesbetweeneubacteriaandeukaryotesBacteriaRibosome:30S+50S->70SFewinitiationfactors:IF-1(eIF1A),IF-2(eIF5B),IF-3(?)ElongationfactorsEF1A(EF-Tu),EF1B(EF-Ts),EF2(EF-G)ReleasefactorsRF-1,RF2,RF3RibosomerecyclingfactorRRFmRNAisnotcappedDirectbindingof30Sparticlenexttoinitiationcodon(AUG)atShine-Dalgarnosequence,5’-AGGAGGU-3’TranslationcoupledtotranscriptionEukaryotesRibosome:40S+60S->80SManyinitiationfactorseIF1,eIF1A,eIF2,eIF2B,eIF3,eIF4A,eIF4B,eIF4E,eIF4F,eIF4G,eIF4H,eIF5,eIF5B,eIF6ElongationfactorseEF1,eEF2ReleasefactorseRF1,eRF3MostmRNAiscappedat5’endandpolyadenylatedat3’end40Sparticleisrecruitedto5’capstructureorpoly(A)tailoraninternalribosomeentrysite(IRES)Translationinalways(?)incytoplasmapartfromtranscriptiontRNAUpto50(eukaryotes),or30-35(bacteria)differenttRNAsCloverleafstructureUnusualbases-covalentmodificationaftertranscriptionbutbeforetRNAsleavenucleusAcceptorarm:7basepairsfollowedbyxCCA-3’aaattachedto2’or3’-OHofterminalAbyClassIandClassIIaa-tRNAsynthetases,respectivelyTYCarmformsonecontinuoushelixwithacceptorarmDarm(dihydro-uridine)interactswithTYCloopviaunusualH-bondsVloopshortinClassItRNAs,longinClassII.AnticodonarmcontainsbasetripletthatpairswithmRNA

codon3’ACCanticodonloop5’TYCloopacceptorarmDloopVloop3’5’TYCDVAnticodonarmAminoacyl-tRNAsynthetasesSynthetaseattachesaatotRNAinatwo-stepprocess:adenylationofaa20aa-tRNAsynthetases,oneforeachaa.Bacteriahaveoftenfewersynthetases,andonesynthetaseattachesdifferentaminoacidstotRNA.AnotherenzymethenchemicallymodifiestheincorrectlyattachedaasothatitcorrespondstotheanticodonofthetRNATwoclassesofaa-tRNAsynthetasesClassIbindsminorgrooveofacceptorarm,ClassIIbindsmajorgrooveofacceptorarm(therearenewlyfoundexceptions)aa-tRNAsynthetaseshavebeenengineeredtoincorporateunusualaminoacids(P.Schultz,S.Yokoyama)Asp-tRNA-synthetaseaa-tRNAsynthaseComplexofClassITyr-tRNA

synthetasewithtRNAtyr

Fig.3.Interactionsbetweentyrosyl-tRNAsynthetaseandtRNAtyr.(A)TheC-terminaldomain(orange)bindsintheelbowbetweenthelongvariablearmandtheanti-codonstemofthetRNA(redbackbone,greenbases).Theanti-codonstemloopinteractswithboththeC-terminaldomainandthe-helicaldomain(pink).ThetRNAmakesnocontactwiththecatalyticdomainofthesamesubunit(cyan).(B)Theunusualconformationoftheanti-codontripletinwhichAde-36isstackedonGua-34,whilePsu-35bulgesout.(C)Base-specificinteractionsofAsp-259fromthe-helicaldomainwithGua-34andAsp-423fromtheC-terminaldomainwithPsu-35.Tyr-tRNAsynthasecomplexwithtRNATyrmRNALinearinbacteria-cancircularizeineukaryotes(viaPabp,eIF4GandeIF4E)Inbacteria,ribosomeisrecruitedtoAUGcodonviaaShine-Dalgarnosequence5’AGGAGGU-(X)3-10-AUG3’Ineukaryotes,mRNAisusuallycappedandpoly-adenylated-aconsensussequenceisfoundaroundtheinitiationcodon-ACCAUGG-(Kozaksequence)5’end-5’UTR-AUG-codingregion-stopcodon-3’UTR-poly(A)tailCappedmRNACappinghappensrightaftertranscription,afterabout25nucleotideshavebeensynthesizedCappingbythreeenzymes:Phosphataseremovesonephosphatefrom5’endGuanyltransferaseaddsaGMPinreverselinkage(5’to5’insteadof5’to3’)MethyltransferaseaddsamethyltotheguanosineSomeRNAsarealsomethylatedatthesecondnucleotideAllthreeenzymesbindtothephosphorylatedRNApolymerasetailCapadditiondistinguishesmRNAfromotherRNAsandhelpstodirecttheribosometomRNACapisrecognizedbythecap-bindingcomplex(CBC),consistingoftwoproteins,CBP80andCBP20.CapisstackedbetweentwotyrosinesY20andY43ofCBP20.Bindinisachievedviathep-stackingeffect.CBCstabilizesthemRNAandinteractswithnuclearporecomplexduringexportofmRNA.Incytoplasm,CBCisreplacedwitheIF4EthathelpstorecruittheribosometomRNA.Herethem7Gisstackedbetweentwotryptophanes(p-stacking)PolyadenylationofmRNA-bindingofPabpandotherfactors3’-endispolyadenylatedbyCstF(cleavagestimulatingfactor),CPSF(cleavageandpolyadenylationspecificityfactor)andPAP(poly(A)polymerase).Poly(A)tailbindsmultiplecopiesofPabp(poly(A)-bindingprotein)OtherfactorsbindmRNA,SRproteins,hnRNPsetc.bindtomRNAandmakeitreadyforexportSomebutnotalloftheattachedproteins(CBC,Pabp)areexportedwiththemRNAIncytosole,CBCisreplacedwitheIF4Efortranslation.mRNAstructureEukaryoticmRNAcancircularizebycouplingthecap-bindingprotieneIF4EandPabptothescaffoldproteineIF4G5’UTRsometimescontainslongGC-richregionsthattendtoformsecondarystructureandinhibitribosomescanning.ThisisfoundparticularlyinmRNAsforgrowth-promotingproteins(growthfactors,oncogeneproducts)andisthoughttobearegulatoryelementtopreventuncontrolledcellgrowth.SomemRNAscontainsecondarystructuresthatallowfordirectbindingofthesmallribosomalparticle,aidedbysegmentsofeIF4G.Thisiscalledaninternalribosomeentrysite,IRES.m7GpppN--eIF4EeIF4GPabpAAAAAAAAAUGGeneticCodeandCodonUsageGeneticCodeisdegenerate.64codons20aminoacids<50tRNAs20synthetasesWobblebasepairing:insometRNAs,3rdbaseofanticodoncanpairwithdifferentbasesofcodonsCommonaahavemultiplecodonsandmultipletRNAs,rarestaminoacids,trpandmetareeachencodedbyonlyonecodonCodonusagefrequencyvarieswithorganism.Canbelookedupat: http://www.kazusa.or.jp/codon/ImportantforexpressionofmammalianproteinsinE.coli.Optimizecodonsforexpression.Escherichiacoli[gbbct]:11985CDS's(3688954codons)fields:[triplet][aminoacid][fraction][frequency:perthousand]([number])UUUF0.5822.2(81958)UCUS0.1710.4(38427)UAUY0.5917.5(64717)UGUC0.465.2(19357)UUCF0.4216.0(59150)UCCS0.159.1(33697)UACY0.4112.2(44909)UGCC0.546.1(22348)UUAL0.1414.4(53048)UCAS0.149.0(33177)UAA*0.612.0(7408)UGA*0.301.0(3684)UUGL0.1313.0(47827)UCGS0.148.5(31383)UAG*0.080.3(996)UGGW1.0013.9(51416)CUUL0.1211.9(43948)CCUP0.187.5(27601)CAUH0.5812.5(46295)CGUR0.3619.9(73524)CUCL0.1010.2(37561)CCCP0.135.4(19840)CACH0.429.3(34207)CGCR0.3619.6(72420)CUAL0.044.2(15655)CCAP0.208.6(31840)CAAQ0.3414.6(53879)CGAR0.073.8(13999)CUGL0.4748.2(177820)CCGP0.4920.8(76842)CAGQ0.6628.4(104717)CGGR0.115.9(21773)AUUI0.4929.8(109873)ACUT0.1910.4(38312)AAUN0.4920.7(76457)AGUS0.169.9(36590)AUCI0.3923.6(87131)ACCT0.4021.9(80904)AACN0.5121.4(78873)AGCS0.2415.1(55819)AUAI0.127.0(25709)ACAT0.179.4(34580)AAAK0.7435.3(130185)AGAR0.073.7(13500)AUGM1.0026.4(97325)ACGT0.2513.7(50690)AAGK0.2612.5(45938)AGGR0.042.1(7787)GUUV0.2819.8(73179)GCUA0.1817.1(62923)GAUD0.6332.8(120820)GGUG0.3525.4(93737)GUCV0.2014.3(52706)GCCA0.2624.2(89153)GACD0.3719.2(70721)GGCG0.3727.0(99602)GUAV0.1711.6(42768)GCAA0.2321.2(78120)GAAE0.6839.0(144050)GGAG0.139.6(35295)GUGV0.3524.3(89623)GCGA0.3230.0(110528)GAGE0.3218.7(68998)GGGG0.1511.3(41635)Homosapiens[gbpri]:50031CDS's(21930294codons)fields:[triplet][aminoacid][fraction][frequency:perthousand]([number])UUUF0.4617.1(374332)UCUS0.1814.7(323470)UAUY0.4412.1(264652)UGUC0.4510.1(221863)UUCF0.5420.4(448127)UCCS0.2217.5(384476)UACY0.5615.5(339473)UGCC0.5512.4(271056)UUAL0.077.3(160731)UCAS0.1511.9(260418)UAA*0.280.8(16884)UGA*0.501.4(30111)UUGL0.1312.7(277774)UCGS0.064.5(98166)UAG*0.220.6(12911)UGGW1.0013.0(284246)CUUL0.1312.9(283480)CCUP0.2817.3(380219)CAUH0.4110.6(231860)CGUR0.084.7(102673)CUCL0.2019.5(428574)CCCP0.3320.0(439256)CACH0.5915.0(329569)CGCR0.1910.8(236986)CUAL0.077.0(153837)CCAP0.2716.7(367297)CAAQ0.2611.9(261063)CGAR0.116.3(138297)CUGL0.4040.1(880072)CCGP0.117.0(154028)CAGQ0.7434.4(755209)CGGR0.2111.8(257761)AUUI0.3615.8(346233)ACUT0.2412.9(283671)AAUN0.4616.7(365457)AGUS0.1512.0(263279)AUCI0.4821.3(466577)ACCT0.3619.1(419213)AACN0.5419.3(422697)AGCS0.2419.4(424788)AUAI0.167.2(157385)ACAT0.2814.9(325763)AAAK0.4224.0(526117)AGAR0.2111.7(255681)AUGM1.0022.3(489160)ACGT0.126.2(135294)AAGK0.5832.5(713826)AGGR0.2011.6(254743)GUUV0.1810.9(239795)GCUA0.2618.6(408931)GAUD0.4622.1(484271)GGUG0.1610.8(237026)GUCV0.2414.6(320190)GCCA0.4028.4(622538)GACD0.5425.7(563848)GGCG0.3422.6(495700)GUAV0.117.0(154102)GCAA0.2316.0(350382)GAAE0.4229.0(634985)GGAG0.2516.4(358824)GUGV0.4728.7(630151)GCGA0.117.6(165700)GAGE0.5840.3(884368)GGGG0.2516.4(360728)Ribosomes70S(2.5M)80S(4.2M)50S(1.6M)30S(0.9M)60S(2.8M)40S(1.4M)5SrRNA(120nt)23SrRNA(2900nt)34proteins16SrRNA(1540nt)21proteins5SrRNA(120nt)28SrRNA(4700nt)5.8SrRNA(160nt)~49proteins18SrRNA(1900nt)33proteinsProkaryotesEukaryotesDistinctrolesofribosomalparticlesSmallparticlegatherscomponents,f-Met-tRNAMet,mRNA,initiationfactors;iscrucialfordecodingSomeantibiotics(streptomycin)interferewithdecodingprocessLargeparticlejoinsaftercomponentshavebeenassembledandperformsproteinsynthesis,inthepresenceofsmallparticle.Largeparticleistargetofmacrolideantibiotics(erythromycinetc.)RibosomestructureRibosomewasamainfocusofstructuralbiologists,andstructurewassolvedina30-yeareffortX-raycrystallographyYonath(30Sand50Sofeubacterium,worksince1980)Schluenzenetal.Cell,102,615(2000)30Sparticleat3.3ÅresolutionHarmsetal.,Cell107,679(2002)50Sparticleat3.1ÅresolutionSteitz&Moore(50S)Banetal.Cell,93,1105–1115,(1998)9ÅresolutionBanetal.Nature,400,841-(1999)5ÅresolutionBanetal.Science289,905-920(2000)2.4ÅresolutionNissenetal.Science289,920-930(2000)RibosomeactivityRamakrishnan(30S)Clemonsetal.,Nature,400,833(1999)5.5ÅCarteretal.Science,291,498(2001)3.1ÅstructurewithIF1boundNoller(70S)Cateetal.,Science2852095-2104(1999)7.8ÅYusupovetal.Science292,883-896(2001)5.5ÅCryo-EMAgarwaletal.,PNAS95,6134(1988)Starketal.Cell100,301(2000)50Sparticleofbacterialribosome(Steitz&Moore)5Åresolutionstructure23SRNA(2900nucleotides),5SRNA(120nucleotides),33proteinsLocationofsomeoftheproteinsDeepactivesitecleftAcceptorarmsofaa-tRNAsmustdiveintocleftExittunnelatthebottomofthelargeactive-sitecleft(initialevidencefromworkofUnwinetal.,1986,andYonathandWittman,1987PositioningofelongationfactorEF-GCentralprotuberance(CP),L1protein,crownviewBanetal.,Nature400,841(1999)Mappingofthetunnelwithtungstenclusters50Sparticle

AllRNAandproteinsdefinedCatalyticcleftincenterExittunnelBanetal.Cell,93,1105–1115,(1998)

9ÅresolutionBanetal.Nature,400,841(1999)5ÅresolutionBanetal.Science289,905(2000)2.4Åresolution30Sparticle

(Ramakrishnan)Clemonsetal