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1、J.Mol.Biol.(1992)227,463-479MutationalAnalysisofthe“Slippery-sequence”ComponentofaCoronavirusRibosomalFrameshiftingSignalIanBrierley-f,AlisonJ.JennerDivisionofVirology,DepartmentofPathology,UniversityofCambridgeTe,nnisCourtRoad,Cambridge.CB2l&P,U.K.andStephenC.InglisCantabPharmaceuticalsResea
2、rchLimited,184CambridgeSciencePark,MiltonRocrdCambridge,CB44GN,I’.K.(Received18February1992;accepted29May1992)TheribosomalframeshiftsignalinthegenomicRNAofthecoronavirusIBViscomposedoftwoelements,aheptanucleotide“slippery-sequence”andadownstreamRNApseudoknot.Wehaveinvestigatedthekindsofslippe
3、rysequencethatcanfunctionattheIBVframeshiftsitebyanalysingtheframeshiftingpropertiesofaseriesofslippery-sequencemutants.WefirstlyconfirmedthatthesiteofframeshiftinginTBVwasattheheptanucleotidest’retchUUCAAAC,andthenusedourknowledgeoft,hepseudoknotstructureandasuitablereportergenetoprepareanex
4、pressionconstructthatallowedboththemagnitudeanddirectionofribosomalframeshiftingtobedeterminedforcandidateslipperysequences.Ourresultsshowthatinalmostallofthesequencestested,frameshiftingisstrictlyintothe-1readingframe.Monotonousrunsofnucleotides,however,gavedetectablelevelsofa-21+1frameshift
5、.product,andUstretchesinparticulargavesignificantlevels(2%t’o%I?/,).PreliminaryevidencesuggeststhattheRN’,4pseudoknotmayplayaroleininfluencingframeshiftdirection.Thespectrumofslip-sequencestestedinthisanalysisincludedallt,hoseknownorsuspectedtobeutilizedinuivo.Ourresultsindicatethattripletsof
6、A,C,GandL’arefunctionalwhendecodedintheribosomalP-sitefollowingslippage(XXXYYYN)although(Jtripletsweretheleasteffective.IntheA-site(XXYmN),tripletsofCandGwerenon-functional.Theidentityofthenucleotideatposition7oftheslipperysequence(XXXYYYN)wasfoundtobeacriticaldeterminantofframeshiftefficienc
7、yandweshowthatahierarchyofframeshiftingexistsfor,4-siterodons.Theseobservationsleadust’osuggestthatribosomalframeshiftingataparticularsiteisdetermined,atleast’inpart.b2thestrengthoftheinteractionofnormalcellulartRNAswit,htheA-sitecodonanddoes
