Translation 翻译.ppt

EukaryoticmRNATranscriptsareProcessed A Simple EukaryoticGene Transcription ProcessingofeukaryoticmRNA RNAprocessingachievesthreethings 1 RemovalofIntrons2 Additionofa5 cap3 Additionofa3 tailThemRNAthenmovesoutofthenucleusandistranslatedinthecytoplasm Translationalcontrol StructureofeukaryoticandprokaryoticmRNAs Modelofeukaryoticribosome rRNAsarebelievedtoplayacatalyticroleinproteinsynthesis Afterremovalof95 oftheribosomalproteins the60Ssubunitcancatalyzeformationofpeptidebonds RibosomalproteinsarenowbelievedtohelpfoldtherRNAsproperlyandtopositionthetRNAs Small largeribosomalsubunits ABindingsiteforthemRNAispresentonsmallsubunit Twobindingsites P A bindtRNAsonlargesubunit Psite holdsthetRNAcarryingthegrowingpolypeptidechain Asite holdsthetRNAwiththenextAAtobeadded RibosomesholdthemRNAandtRNAstogetherandconnecttheaminoacidsattheAsitetothegrowingpolypeptide RibosomeStructure StructureoftRNAAlignseachaminoacidwiththecorrespondingcodon 70 80ntlong 3 endhasthe5 CCAsequencetowhichaaarelinked Theoppositeendcontainstheanticodonloop Containsmodifiedbases ManyRNAViruseshavecappedgenomicRNAssimilartoeukaryotichostmRNAs MosteukaryoticmRNAsarecappedatthe5 endduringnuclearprocessing Theterminal5 phosphateisfirstremovedbya5 triphosphatase GuanyltransferasetransfersGMPfromGTPtothe5 endofthemRNAtoaddtheGpppNcapstructure The5 terminalinvertedGresidueisthenmodifiedbymethylation ManyRNAvirusesreplicateinthecytoplasmandmustuseaviraldependentcappingmechanismsuppliedbytheRNA Dependent RNAPolymerase TheCapstructure m7GpppN ismostcommoninviralandmammalianmRNAs ThreedistinctstagesoftranslationInitiation Ratelimitingstep RequireshydrolysisofATPandGTP ResultsinformationofacomplexcontainingthemRNA theribosomeandtheinitiatorMet tRNAA 5 end Cap dependentinitiation Theinitiationcomplexbindstothe5 capstructureandscansina5 to3 directionuntilinitiatingAUGisencounteredB InternalribosomeentryInitiationcomplexbindsupstreamofinitiationcodon Thefirststepistherecognitionofthe5 capbyeIF4F whichconsistsofthreeproteins eIF4E eIF4GandeIF4A Capbindingprotein eIF4E bindstocap TheN terminusofeIF4GbindseIF4EandtheC terminusbindseIF4A The40SsubunitbindstoeIF4GviaeIF3 5 end cap dependentinitiation The40Scomplexthenscansdownthe5 untranslatedregiontothefirstAUGcodon AGTPhydrolysisstepbyeIF5triggersGDPbindingofeIF2andreleaseofinitiationproteins The60Ssubunitjoinsthecomplexandthe80SribosomeinitiatestranslatetheORF Aninitiationcomplexformsatthecapwiththe40Sribosomalsubunitandothertranslationinitiationfactors Cap DependentInitiationofProteinSynthesisinEukaryotes RibosomeselectsaminoacylatedtRNAeEF1aandGTPareboundtoaminoacylatedtRNARibosomecatalyzesformationofapeptidebondTranslocationisdependentoneEF2andGTPhydrolysisManyribosomesmaytranslatemRNAssimultaneouslyonthesamestrand Elongation PA eEF1a eEF2 GTP GTP TerminationTranslationisterminatedatoneofthreestopcodons UAA UAG UGA TerminationcodonattheAsiteisrecognizedbythereleasefactorinsteadofatRNA Thereleasefactorbindstheterminationcodon ThepeptidechainisthenreleasedfollowedbydissociationofthetRNAandtheribosome The5 enddependentinitiationisstimulatedbythepoly A bindingproteinPabp1p whichinteractswitheIF4G ThisinteractioncircularizesthemRNAandfacilitatesformationoftheinitiationcomplexMechanismtoensurethatonlyintactmRNAistranslated Closedloopmodel mRNAsofpicornaviruseslack5 capRibosomesbindinternallyratherthanatthemRNA5 end5 endofpoliovirusRNApromotesinternalbindingof40Ssubunitatinternalribosomeentrysite IRES InpoliovirusinfectedcellseIF4Giscleaved inactivatingtranslationofcellularmRNAsTheinitiationintheIRESdoesnotdependonthepresenceofacapstructure butrequiresC terminalfragmentofeIF4Gtorecruitthe40SsubunitthroughinteractionwitheIF3 5 end cap independentinitiation Poliovirus FivedifferenttypesofIRESsequencesarefoundonviralRNAs TypeI enteroandrhinoviruses poliovirus Initiationcodonislocatedpastthe3 endoftheIRES40SbindstoIRESscanstoAUGTypeII cardioandapthoviruses EMCV Initiationcodonisatthe3 endoftheIRES40SbindsatornearAUGnoscanningoccursTypeIII hepatitisAvirusinitiationcodonislocatedpastthe3 endoftheIRESrequiresallofinitiationproteins includingeIF4ETypeIV hepatitisCvirusThe3 endofthehepatitisCvirusIRESextendsbeyondtheAUGcodonTypeV cricketparalysisvirusIRESendsattheinitiationcodon althoughitisnotanAUGcodon noinitiationfactorsarerequiredinitiationcodonisplacedattheAsiteinsteadofthePsite FourtypesofIRES polivirus Encephelomyocarditisvirus EMCV HepatitisC Cricketparalysisvirus HepatitisCvirusIRES 40SribosomalsubunitbindsdirectlytoHepatitisCvirusIRESintheabsenceofmostinitiationfactorsAdramaticchangeintheconformationofthe40SsubunitoccurswhenitbindsHepatitisCvirusIRESsettingtheAUGatthePsiteeIF3alsobindstotheHepatitisCvirusIRES BicistronicmRNAassayforIRESelements Viraltranslationstrategies PolyproteinsynthesisPicornaviruses Entire senseRNAgenomeistranslatedintoasinglelargepolyprotein Processingiscarriedoutbytwovirusencodedproteases2Aproand3Cpro Flaviviruses Viralprecursorproteinsareprocessedbycellularproteases The senseRNAgenomeistranslatedintoapolyproteinprecursorprocessedbyviralserineproteaseandbyhostsignalpeptidase Potyvirusgroupofplantviruses PotatovirusYandtobaccoetchviruscontaina sensegenomeRNAofaround10 000baseswhichhasasingleopenreadingframe Thispolyproteinisprocessedbyviralencodedproteases Polyproteinprocessinginenterovirusesandflaviviruses Poliovirus Flavivirus LeakyscanningAlthoughmajorityofeukaryoticmRNAsaremonocistronic someviralmRNAsencodeoverlappingreadingframes Thefirststartsiteisinapoorcontext someribosomescanbypassitandinitiateatthesecondAUG whichhasabettercontext Thiswillresultintranslationoftwodifferentproteins ReinitiationRareineukaryotes butverycommoninprokaryoticcellularandviralmRNAs SomeeukaryoticmRNAscontainupstreamAUGcodonsthatterminatebeforethedownstreamreadingframe Theupstreamopenreadingframesmaybetranslated withreinitiationoccurringatthedownstreamopenreadingframe IninfluenzaBvirusmRNA M2initiationcodonispartoftheterminationcodonforM1protein M2synthesisisnotefficientanddependentonM1synthesis ReinitiationoftranslationininfluenzaBvirus SuppressionofterminationSuppressionofterminationoccursduringtranslationofmayviralmRNAsasameansofgeneratingasecondproteinwithextendedcarboxyterminus Inretroviruses gagandpolgenesareencodedbyasinglemRNAandseparatedbyanamberterminationcodonUAG Translationalsuppressionoftheambercodonallowssynthesisofthegagpolprecursor Asimilarstrategyisusedbytobaccomosaicvirustotranslateitsreplicaseproteins TranslationsuppressionismediatedbysuppressortRNAsthatcanrecognizeterminationcodonsandinsertaspecificaminoacid Thenucleotidesequence3 oftheterminationcodonalsoplaysanimportantroleintheefficiencyoftranslationalsuppression Suppressionofterminationcodonsinalphavirusesandretroviruses Pseudonot alphavirus retrovirus RibosomalframeshiftingRibosomalframeshiftingisaprocessinwhichribosomesmovetoadifferentreadingframeandcontinuetranslationinthatreadingframe ItwasdiscoveredincellsinfectedwithRoussarcomavirusandhassincebeendescribedformanyothervirusesincludingotherretroviruses strandRNAvirusesandherpessimplexvirus Requiresa slippery sequenceX XXY YYZ inRoussarcomavirusA AAU UUA andanRNAsecondarystructurecalledapseudoknotfivetoeightnucleotidesdownstream TwotRNAsinthezeroreadingframeslipbackbyonenucleotidetothe 1phaseandeachtRNAbasepairswiththemRNAinthefirsttwonucleotidesofeachcodon AsaresultoftheframeshiftaGag polfusionisproducedatabout5 ofthelevelofGagprotein Ribosomalframeshifting RoussarcomavirusmRNAencodesGagandPolproteinsthatoverlapina 1readingframe REGULATIONOFTRANSLATIONDURINGVIRALINFECTIONInterferonsareproducedinresponsetoviralinfectionaspartoftherapidinnateimmuneresponseIn