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DNACytoplasmNucleusEukaryoticmRNATranscriptsareProcessedExportGAAAAAARNATranscriptionNuclearporesGAAAAAARNAProcessingmRNAA“Simple”EukaryoticGeneTerminatorSequencePromoter/ControlRegionTranscriptionStartSite5’UTR3’UTRExonsIntrons3’5’Exon2Exon3Int.2Exon1Int.13’5’Exon2Exon3Exon1Int.2Int.1TranscriptionUnprocessedRNATranscriptProcessingofeukaryoticmRNARNAprocessingachievesthreethings:1)RemovalofIntrons2)Additionofa5’cap3)Additionofa3’tailThemRNAthenmovesoutofthenucleusandistranslatedinthecytoplasm.3’PolyATailProteinCodingRegion

3’UTR

5’UTRG5’CapExon2Exon3Exon1Int.2Int.1AAAAATranslationalcontrolStructureofeukaryoticandprokaryoticmRNAs:Modelofeukaryoticribosome?rRNAsarebelievedtoplayacatalyticroleinproteinsynthesis.?

Afterremovalof95%oftheribosomalproteins,the60Ssubunitcancatalyzeformationofpeptidebonds.?RibosomalproteinsarenowbelievedtohelpfoldtherRNAsproperlyandtopositionthetRNAs.Small&largeribosomalsubunits.ABindingsitefor themRNAispresentonsmallsubunit.Twobindingsites (P&A)bindtRNAsonlargesubunit.Psite–holdsthetRNAcarryingthegrowingpolypeptidechain.Asite–holdsthetRNAwiththenextAAtobeadded.

RibosomesholdthemRNAandtRNAstogetherandconnecttheaminoacidsattheAsitetothegrowingpolypeptide.RibosomeStructureStructureoftRNA

Alignseachaminoacidwiththecorrespondingcodon?70-80ntlong?3’endhasthe5’-CCAsequencetowhichaaarelinked?Theoppositeendcontainstheanticodonloop?

Containsmodifiedbases

ManyRNAViruseshavecappedgenomicRNAssimilartoeukaryotichostmRNAs?MosteukaryoticmRNAsarecappedat the5’endduringnuclearprocessing.?Theterminal5’phosphateisfirst removedbya5’triphosphatase.?GuanyltransferasetransfersGMPfrom GTPtothe5’endofthemRNAtoadd theGpppNcapstructure.?The5’terminalinvertedGresidueis thenmodifiedbymethylation.?ManyRNAvirusesreplicateinthe cytoplasmandmustuseaviral dependentcappingmechanism suppliedbytheRNA-Dependent-RNA Polymerase.?TheCapstructure,m7GpppN,ismost commoninviralandmammalian mRNAs.Threedistinctstagesoftranslation

Initiation

?Ratelimitingstep

?RequireshydrolysisofATPandGTP

?ResultsinformationofacomplexcontainingthemRNA,theribosomeandtheinitiatorMet-tRNA

A.

5’end(Cap)dependentinitiation?Theinitiationcomplexbindstothe5’capstructureandscansina5’to3’directionuntilinitiatingAUGisencounteredB.Internalribosomeentry

Initiationcomplexbindsupstreamofinitiationcodon?Thefirststepistherecognitionofthe5’capbyeIF4F,whichconsistsofthreeproteins,eIF4E,eIF4GandeIF4A.Capbindingprotein,eIF4E,bindstocap?TheN-terminusofeIF4GbindseIF4EandtheC-terminusbindseIF4A?The40SsubunitbindstoeIF4GviaeIF35’end(cap)dependentinitiation:The40Scomplexthenscansdownthe5’untranslatedregiontothefirstAUGcodon.AGTPhydrolysisstepbyeIF5triggersGDPbindingofeIF2andreleaseofinitiationproteins.The60Ssubunitjoinsthecomplexandthe80SribosomeinitiatestranslatetheORF.Aninitiationcomplexformsatthecapwiththe40Sribosomalsubunitandothertranslationinitiationfactors.Cap-DependentInitiationofProteinSynthesisinEukaryotesRibosomeselectsaminoacylatedtRNAeEF1aandGTPareboundtoaminoacylatedtRNARibosomecatalyzesformationofapeptidebondTranslocationisdependentoneEF2andGTPhydrolysisManyribosomesmaytranslatemRNAssimultaneouslyonthesamestrand.ElongationPAeEF1aeEF2GTPGTPTerminationTranslationisterminatedatoneofthreestopcodons(UAA,UAG&UGA).

?TerminationcodonattheAsiteisrecognizedbythereleasefactorinsteadofatRNA?Thereleasefactorbindstheterminationcodon?ThepeptidechainisthenreleasedfollowedbydissociationofthetRNAandtheribosome?The5’enddependentinitiationisstimulatedbythepoly(A)bindingproteinPabp1p,whichinteractswitheIF4G?ThisinteractioncircularizesthemRNAandfacilitatesformationoftheinitiationcomplexMechanismtoensurethatonlyintactmRNAistranslatedClosedloopmodel:?mRNAsofpicornaviruseslack5’capRibosomesbindinternallyratherthanatthemRNA5’end5’endofpoliovirusRNApromotesinternalbindingof40Ssubunitatinternalribosomeentrysite(IRES)InpoliovirusinfectedcellseIF4Giscleaved,inactivatingtranslationofcellularmRNAsTheinitiationintheIRESdoesnotdependonthepresenceofacapstructure,butrequiresC-terminalfragmentofeIF4Gtorecruitthe40SsubunitthroughinteractionwitheIF3.5’end(cap)independentinitiation:PoliovirusFivedifferenttypesofIRESsequencesarefoundonviralRNAs:

TypeI-enteroandrhinoviruses(poliovirus) Initiationcodonislocatedpastthe3’endoftheIRES

40SbindstoIRESscanstoAUGTypeII-cardioandapthoviruses(EMCV) Initiationcodonisatthe3’endoftheIRES 40SbindsatornearAUGnoscanningoccursTypeIII-hepatitisAvirus initiationcodonislocatedpastthe3’endoftheIRES requiresallofinitiationproteins,includingeIF4ETypeIV-hepatitisCvirus The3’endofthehepatitisCvirusIRESextends beyondtheAUGcodonTypeV-cricketparalysisvirus

IRESendsattheinitiationcodon,althoughitisnotan AUGcodon,noinitiationfactorsarerequired initiationcodonisplacedattheAsiteinsteadoftheP siteFourtypesofIRES:polivirusEncephelomyocarditisvirus(EMCV)HepatitisCCricketparalysisvirusHepatitisCvirusIRES:40SribosomalsubunitbindsdirectlytoHepatitisCvirusIRESintheabsenceofmostinitiationfactorsAdramaticchangeintheconformationofthe40SsubunitoccurswhenitbindsHepatitisCvirusIRESsettingtheAUGatthePsiteeIF3alsobindstotheHepatitisCvirusIRESBicistronicmRNAassayforIRESelementsViraltranslationstrategiesPolyproteinsynthesis

Picornaviruses-Entire(+)senseRNAgenomeistranslatedintoasinglelargepolyprotein.Processingiscarriedoutbytwovirusencodedproteases2Aproand3Cpro.

Flaviviruses-Viralprecursorproteinsareprocessedbycellularproteases.The(+)senseRNAgenomeistranslatedintoapolyproteinprecursorprocessedbyviralserineproteaseandbyhostsignalpeptidase.

Potyvirusgroupofplantviruses-PotatovirusYandtobaccoetchviruscontaina(+)sensegenomeRNAofaround10,000baseswhichhasasingleopenreadingframe.Thispolyproteinisprocessedbyviralencodedproteases.PolyproteinprocessinginenterovirusesandflavivirusesPoliovirus:Flavivirus:

Leakyscanning

AlthoughmajorityofeukaryoticmRNAsaremonocistronic,someviralmRNAsencodeoverlappingreadingframes.Thefirststartsiteisinapoorcontext,someribosomescanbypassitandinitiateatthesecondAUG,whichhasabettercontext.Thiswillresultintranslationoftwodifferentproteins.Reinitiation

Rareineukaryotes,butverycommoninprokaryoticcellularandviralmRNAs.SomeeukaryoticmRNAscontainupstreamAUGcodonsthatterminatebeforethedownstreamreadingframe.Theupstreamopenreadingframesmaybetranslated,withreinitiationoccurringatthedownstreamopenreadingframe.

IninfluenzaBvirusmRNA,M2initiationcodonispartoftheterminationcodonforM1protein.M2synthesisisnotefficientanddependentonM1synthesisReinitiationoftranslationininfluenzaBvirusSuppressionoftermination

SuppressionofterminationoccursduringtranslationofmayviralmRNAsasameansofgeneratingasecondproteinwithextendedcarboxyterminus.Inretroviruses,gagandpolgenesareencodedbyasinglemRNAandseparatedbyanamberterminationcodonUAG.Translationalsuppressionoftheambercodonallowssynthesisofthegagpolprecursor.

Asimilarstrategyisusedbytobaccomosaicvirustotranslateitsreplicaseproteins.

TranslationsuppressionismediatedbysuppressortRNAsthatcanrecognizeterminationcodonsandinsertaspecificaminoacid.Thenucleotidesequence3’oftheterminationcodonalsoplaysanimportantroleintheefficiencyoftranslationalsuppression.SuppressionofterminationcodonsinalphavirusesandretrovirusesPseudonotalphavirusretrovirusRibosomalframeshifting

Ribosomalframeshiftingisaprocessinwhichribosomesmovetoadifferentreadingframeandcontinuetranslationinthatreadingframe.

ItwasdiscoveredincellsinfectedwithRoussarcomavirusandhassincebeendescribedformanyothervirusesincludingotherretroviruses,(+)strandRNAvirusesandherpessimplexvirus.

Requiresa“slippery”sequenceX-XXY-YYZ(inRoussarcomavirusA-AAU-UUA)andanRNAsecondarystructurecalledapseudoknotfivetoeightnucleotidesdownstream.

TwotRNAsinthezeroreadingframeslipbackbyonenucleotidetothe–1phaseandeachtRNAbasepairswiththemRNAinthefirsttwonucleotidesofeachcodon.

AsaresultoftheframeshiftaGag-polfusionisproducedatabout5%ofthelevelofGagprotein.

RibosomalframeshiftingRoussarcomavirusmRNAencodesGagandPolproteinsthatoverlapina–1readingframe

REGULATIONOFTRANSLATIONDURINGVIRALINFECTION

Interferonsareproducedinresponsetoviralinfectionaspartoftherapidinnateimmune

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