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1、Proc.Natl.Acad.Sci.USAVol.84,pp.4762-4766,July1987BiochemistrySpecializedribosomesystem:PreferentialtranslationofasinglemRNAspeciesbyasubpopulationofmutatedribosomesinEscherichiacoli(Shine-Dalgarnosequence/rrnBoperon)ANNAHuIANDHERMANA.DEBOER*DepartmentofCellGenetics,Genentech,Inc.,460PointSanBrunoBo
2、ulevard,SouthSanFrancisco,CA94080CommunicatedbyHerbertW.Boyer,March6,1987(receivedforreviewNovember2,1986)ABSTRACTInEscherichiacoli,allmRNAsaretranslatedbyonepool offunctionallyidenticalribosomes.Here,wedescribeasysteminwhichasubpopulationofmodifiedribosomesaredirectedtoasinglemutatedmRNAspecies.Thi
3、swasaccomplishedbychangingtheShine-Dalgarnosequencethatprecedestheheterologoushumangrowthhormonegenefrom5GGAGGto5CCTCCor5GTGTG.TranslationofthesemodifiedmRNAsbywild-typeribosomesisveryineffi-cient.Whentheanti-Shine-Dalgarnoregion(i.e.,theregioncomplementarytotheShine-Dalgarnosequence)atthe3endoftheg
4、eneencoding16SrRNA(rrnB)wasalteredfrom5CCTCCto5GGAGGor5CACAC,thusrestoringitspotentialtobase-pairwiththemutatedhumangrowthhor-monemRNA,significantexpressionofthis.mRNAoccurred.Growthhormonesynthesiswasdependentoninductionof-themutatedrrnBoperon.Subsequently,thesespecializedribo-somesweremadespectino
5、mycin-resistaintbytheintroductionofaC-Usubstitutionatposition1192ofthe16SrRNA.Thus,hostproteinsynthesiscouldbeshutoffbytheadditionofspectinomycinandthespecificityandefficiencyofthespecial-izedribosomescouldbeassessed.Sincethespecializedribo-somesrepresentanonessentialsubpopulationinthecell,thissyste
6、moffersanapproachtothestudyofmutationselsewhereinthe16S-rRNAgenethatotherwisewouldbelethaltothecell.TheShine-Dalgarno(SD)sequence(1),whichisgenerallyfound8basesupstreamfromthestartcodoninEscherichiacolimRNAs(2,3),iscomplementarytoaregionclosetothe3endof16SrRNA,hereafterreferredtoastheanti-Shine-Dalg
7、arno(ASD)sequence.ShineandDalgarnopos-tulatedthattheSDsequenceinthemRNAinteractswiththeASDregionof16SrRNAandthatthismRNArRNAbase-pairingplaysanimportantroleintheinitiationofproteinbiosynthesis.Alargebodyofbiochemicalandgeneticevi-dencesupportsthisview(2).Thebase-pairedcomplexescanbeisolatedinvitro(4
8、),andmutationsinthemRNAalteringthecomplementarityhaveaprofoundeffectonitstranslat-ability(5-11).ReplacementoftheSDsequencewithasyntheticDNAfragmentencodinganon-SDsequencees-sentiallyabolishesmRNAtranslation(8,11).TheASDsequenceispartofthehighlyconserved3endofthe16SrRNAfoundinprokaryotes(12).Syntheti
9、coligonucleotideprobescanbeboundspecificallytotheASDregionofthe16SrRNAinintact30Sribosomalsubunits,implyingthatthissequenceisnotcomplexedwithproteinsorRNA(13,14).SinceonepoolofribosomespresumablytranslatesallmRNAs,itisdifficulttoobtainandstudymutationsintherRNAmolecule;mutationsintroducedviasite-dir
10、ectedmu-tagenesisoftenappeartobelethal(15).Inthispaper,wedescribeexperimentsshowingthatamutatedribosomalsubpopulationcanbemadeanddirectedtoasinglemutatedmRNAspecies.ThiswasaccomplishedbychangingtheSDsequenceonthemRNAaswellastheASDsequenceonthe16SrRNAintoentirelydifferentbutcomplementarysequences.Thi
11、ssystemofspecializedribo-somesoffersapowerfulapproachtotheanalysisofadditionalmutationsincrucialregionsoftherRNAmoleculethatotherwisewouldbelethaltothecell.MATERIALSANDMETHODSPlasmidamplificationwasdoneinE.coli294(F-,supE44,endAl,thi-J,hsdR4).Transformationwithplasmidscontain-ingthePLpromoterofbacte
12、riophageXwasdoneinE.coliK5716(F,A(lac-pro),supE,traD36,proAB,lacIQZAM1S),whichisaA+lysogenderivedfromJM101.TemperatureinductionofthespecializedribosomeswithXPLwasdoneinE.coliK5637(cI857,ABamcro27,Oam29).BothK5716andK5637wereconstructedbyH.I.Miller(Genentech).Plasmidconstructionsandblothybridizationa
13、nalysesofelectrophtoreticallyfractionatedRNAweredoneasdescribed(10).NaDodSO4/polyacrylamidegelelectrophoresiswascarriedoutaccordingtotheprocedureofLaemmli(16).RESULTSAssemblyoftheSpecializedRibosomeSystem.InordertoassembleasysteminwhicharibosomalsubpopulationwouldtranslateasinglemRNAspecies,itwasnec
14、essarytoaltertheSDsequenceandtheASDsequenceinsuchawaythatwild-typeribosomeswouldnotrecognizethemutatedmRNAandthatmutatedribosomeswouldnotrecognizethewild-typemRNA.Onewayinwhichthisgoalmightbeaccom-plishedwastoreplacethenaturalSDandASDsequencesbytheircomplementsonboththemRNAandthe16SrRNA.Thus,wechang
15、edtheSDsequencefrom5GGAGGto5CCUCCandtheASDsequencefrom5CCUCCto5GGAGG;thisissystemIX.Sincewewereconcernedthattheseradicalchangesmightimpairribosomalfunctions,wealsoassembledasystemthatdifferedlessradicallyfromthewild-typesystem.InsystemX,theSDsequencewaschangedfrom5GGAGGto5GUGUG.TheASDsequencewaschan
16、gedaccordinglyfrom5CCUCCto5CACAC.InallsystemsthegeneencodingthemutatedmRNAistran-scribedconstitutivelybyapromoterderivedfromthetrppromoter(10),whereasthewild-typeandmutatedrrnBgenesareundercontrolofthe,XPLpromoter(17).Abbreviations:ASDsequence,anti-Shine-Dalgarnosequence;hGH,humangrowthhormone;PSDR,
17、portableShine-Dalgarnoregion;SDsequence,Shine-Dalgarnosequence.*Towhomreprintrequestsshouldbeaddressed.4762Thepublicationcostsofthisarticleweredefrayedinpartbypagechargepayment.Thisarticlemustthereforebeherebymarkedadvertisementinaccordancewith18U.S.C.1734solelytoindicatethisfact.Proc.Natl.Acad.Sci.
18、USA84(1987)4763Adetaileddescriptionoftheconstructionoftheentirespecializedribosomesystemisgivenelsewhere(10).TheessentialfeaturesofthesystemareshowninFig.1,andasummaryofalltheplasmidsusedfortheexperimentsandtheircontrolsisgiveninTable1.InductionoftheMutatedrrnBOperon.SincetheASDregionofthespecialize
19、drRNAdiffersfromthatofthewildtypeatthe3end,itispossibletodistinguishthetwo16SrRNAspeciesbyusingradiolabeledoligodeoxynucleotideprobesthatarespecificforeach.PlasmidsofthepASD-PSDR-PLseriesweretransformedintothehostK5637,whichcarriesageneencodingatemperature-sensitiveXrepressor,andtheaccumulationofthe
20、mutated16SrRNAwasmoni-toredafterinduction.Fig.2showsthatthesyntheticoligo-nucleotideprobes(theirsequencesaregiveninthefigurelegend)thatarecomplementarytoeitherASDIXorXdonothybridizetowild-tSpe16SrRNA(i.e.,torRNAfromcellslackingthespecializedsystem).Thelevelofthemutated16SrRNAincredsesrapidlyandreach
21、esamaximumafterabout1hrofinduction(quantitationdatanotshown).Thesizeofthemutated16SrRNAmoleculesisidenticaltothatofwildtype,suggestingthatthechangesintheASDsequencedonothaveamajoreffectontheprocessingoftherRNAprecursor.ProteinSynthesisbytheSpecializedRibosomes.Fig.3showsthetimecourseofhGHaccumulatio
22、nafterinductionofthespecializedribosomesystems.NotethatthehGHgeneistranscribedconstitutivelyinallfivesystems.AminutePribnowboxPSDRVilI(wt):TAGTTTAATGTGTG(PSDRIX:AHpoIPSDRX:PSIAEcoRIPtrpPvuIK/APRBamHtPASD-FKpn;4PLKpnASDVIII(wt):TTGCASDIX:ASDX:amountofhGHisdetectedwhenspecializedmRNAismadeintheabsence
23、ofspecializedribosomes(asinthecontrolsystemsphGH-PSDRIXand-X).ThehGHmadehereisprobablyduetotheinefficienttranslationofthespecializedmRNAsbythewild-typeribosomes.AlowlevelofhGHisalsosynthesizedinthespecializedsystemsIXandXpriortoinduction.However,uponinductionofPL,thehGHlevelgraduallyincreases.After3
24、hrofinduction,thehGHlevelinsystemXisequalto thatinthewild-typesystem.TheoverallefficiencyofsystemXis2-to3-foldhigherthanthatofsystemIX.Thereasonsforthisdifferenceinefficiencyarenotclear.ThebasecompositionoftheSDsequenceandASDregionmayaffecttherateoftranslationinitiationortherateofelongationdifferent
25、lyinthetwosystems.ThehGH-accumulationdata(Fig.3),alongwiththeRNAdata(Fig.2),showthatthemutated16S-rRNAgeneisindeedtranscribedandthatatleastsomeofthemutatedrRNAmoleculesareprocessedandassembledintofunctionalribosomes.NaDodSO4/polyacrylamidegelelectrophoresisoftotalcellularproteinextractedbeforeorafte
26、rinductionofcellscontainingsystemIXorXrevealstheappearanceofaproteinof22.5kDainbothspecializedsystemscomparelanes2and3(30C)with7and8(370C)inFig.4.ThisproteinhasthesameelectrophoreticmobilityashGH,andimmunoblotanalysisconfirmedthatthisproteinbandisindeedhGH(datanotshown).ThehGHlevelfoundinsystemXisag
27、aingreaterthanthatinsystemIXandiscomparabletothatfoundin+1SDstartGAAGCTTTGGAGGTCTAGAATTCTATG3HindIlCCT0CXbaIEcoRIGTGTGiHIBstEIIASDSATCACCTCCTTA3GGAGGCACACFIG.1.Theplasmid-bornespecializedribosomesystem.Inthissystemthehumangrowthhormone(hGH)geneistranscribedunderthecontrolofaconstitutivemutanttrpprom
28、oter(Ptrp)(10)andthenaturaltrpleaderSDsequence(5AAGG)wasreplacedbythesequence5GGAGGinsystemVIIIbyusingadouble-strandedsyntheticDNAfragment(10).ThesequenceofthemodifiedpromoterandportableSDregion(PSDR)insystemVIII(wild-type,wt)isshown,alongwiththeATGtranslationstartcodon;restrictionendonucleaserecogn
29、itionsitesareunderlined.TheuntranslatedregionofthehGHgeneinsystemsIXandXisthesameasinsystemVIIIexceptfortheSDregion,asshown.Theplasmid-bornerrnBoperonisunderXPLcontrolandistemperature-inducibleduetothepresenceoftheXcI857repressorintheX-lysogenichost.Thesequenceofthe3endofthe16S-rRNAgeneisshownforASD
30、VIII,alongwiththemutatedASDregionsinsystemsIXandX(seeref.10fordetails).Thus,thefinalpASD-PSDR-PLplasmidsencodehGHmRNAwithanSDsequencethatiscomplementarytotheASDsequenceofthemutatedrrnBoperononthesameplasmid.PlasmidseitherlackingthehGHgenepASDVIII(IXandX)-PLorcontainingthehGHgenewithawild-typeSDseque
31、nceandamutatedASDregioninthespecializedribosomes(pASDIX-PSDRVIII-PLandpASDX-PSDRVIII-PL)wereconstructedasexperimentalcontrols(seeTable1).Introductionofanyoftheseplasmidsintothecellsisnotlethal.T1andT2aretranscriptionterminationsites.APRrepresentsthegenethatconfersampicillinresistance.Biochemistry:Hu
32、ianddeBoerDR4764Biochemistry:HuianddeBoerTable1.Summaryoftheplasmidsandsystemsused16S-rRNAASDhGHSDsequencePlasmidsequence(5to3)(5to3)ASD/SDduplexphGH-PSDRVIIIrrnBnotpresentonplasmidGGAGGphGH-PSDRIXrrnBnotpresentonplasmidCCUCCphGH-PSDRXrrnBnotpresentonplasmidGUGUGpASDVIII-PLCCUCC(wildtype)Genenotpres
33、entonplasmidpASDIX-PLGGAGGGenenotpresentonplasmidpASDX-PLCACACGenenotpresentonplasmidpASDVIII-PSDRVIII-PL(systemVIII)CCUCCGGAGG3CCUCC5GGAGGpASDIX-PSDRI-PL(systemIX)GGAGGCCUCC3GGAGG5CCUCCpASDX-PSDRX-PL(systemX)CACACGUGUG3CACAC5GUGUGpASDIX-PSDRVIII(systemIXc)GGAGGGGAGG3GGAGG5GGAGGpASDX-PSDRVIII(system
34、Xc)CACACGGAGG3CACAC5GGAGGpASDVIII-PSDRVIII-PL-SpCr*pASDIX-PSDRIX-PL-SPCrtpASDX-PSDRX-PL-Spcr*Spectinomycin-resistantderivativeofsystemVIII.tSpectinomycin-resistantderivativeofsystemIX.tSpectinomycin-resistantderivativeofsystemX.wild-typecells(comparelanes6and8inFig.4).Thesedataareingeneralagreementw
35、iththekineticdatashowninFig.3.InadditiontothehGHband,anovelproteinof50kDaappearsinsystemIX(lane7)andaproteinof19kDainsystemX(lane8).Apparently,eachkindofspecializedribosomerecognizesasequenceinfrontofalongopenwtIX1rIreadingframethatisnotrecognizedasabindingsiteforwild-typeribosomes.Immunoblotanalysi
36、sshowedthatthe50-kDaproteinisnotantigenicallyrelatedtohGH(datanotshown)andthereforeisnotaread-throughtranslationprod-uctofthehGHmRNA.However,the19-kDaproteinisimmunologicallyrelatedtohGH.However,the19-kDa20wtX1-I16S-*0609006090Time(min)c300(D-10EI-CD0609006090Time(min)FIG.2.Blothybridizationanalysis
37、ofelectrophoreticallyfrac-tionatedRNA,showingtheinductionofrrnBoperonsbearingamutatedASDregion.Cellsweregrownat30Cfor2hr,transferredto42C(timezero)for15min,andthenincubatedat37Cfortheremainingtime.TotalRNAwasisolatedandanalyzedatthevarioustimesafterinductionasdescribed(10).Inthelefttwopanels(wild-ty
38、pecellsandsystemIX),thehybridizationprobespecificforASDIXwasused;intherighttwopanels,theprobeforASDXwasused.ThesequenceoftheASDIXprobeis5TCTTTAAGGTAAC-CTCCTGATCCAACCGCandthatforASDXis5TCTTTAAGGT-AAGTGTGTGATCCAACCGC.0-101Time,hr23FIG.3.hGHaccumulationincellswithspecializedribosomes.Extractionandassay
39、ofhGHwere doneasdescribed(10).A,Wild-typesystemVIII;*,specializedsystemIX;*,specializedsystemX;o,controlsystemphGH-PSDRIX;ando,controlsystemphGH-PSDRX.BothcontrolsystemscontainspecializedmRNAbutlackspecializedribosomes.Valuesarenormalizedtocellculturedensity(ODunit).Proc.Natl.Acad.Sci.USA84(1987)Pro
40、c.Natl.Acad.Sci.USA84(1987)47651234567891092-_Mam66-Mr,.A.i=SOWu45-1W=_=J._.-Nw.0.ArawNNmow*._.-31-_21-*14-i_On_-s_l-uninduced-4-induced-iFIG.4.Proteinprofileofcellscontainingthespecializedsystem,beforeandafterinductionofthespecializedsystem.Forinduction,cellsweregrownat30Cfor2hr,thenshiftedtoa420Cb
41、athfor15min,andfinallytransferredto370Cfor3hr.CellswereharvestedandtheproteinprofilewasanalyzedbyNaDodSO4/12.5%polyacrylam-idegelelectrophoresis;proteinswerevisualizedbyCoomassiebluestain.Lane1representsthewild-typesystem,lanes2and3thespecializedsystemsIXandX,andlanes4and5representthecontrolsystemsp
42、hGH-PSDRVIIIandpASDVIII-PL,allat30C.Lanes6-10representthesamesystemsinthesameorderbutat370C.ThepositionofhGH(22.5kDa)isindicatedbythemiddlearrow.Theupperandlowerarrowsindicatethenovelbands(50kDaand19kDa)thatappearinsystemsIXandX,respectively,at370C.Leftmostlaneshowsstandardproteins,whosemolecularmas
43、sesaregiveninkDa.proteinisimmunologicallyrelatedtohGH.Lane4andlane9(Fig.4)showtheproteinprofileincells(at30Cand370C,respectively)thatmakeawild-typehGHmRNAconstitu-tivelybutlackspecializedribosomes.ThehGHbandispresentatbothtemperatures,asexpected(thehGHmRNAistranslatedbywild-typeribosomes).However,th
44、enovelproteinsof50kDaand19kDaareabsentat370C,indicatingthatthesenovelproteinsasfoundinsystemsIXandXarenotheatshockproteins.Lanes5and10showtheproteinprofilesofnegativecontrolstrainscontainingplasmidsencodingawild-typeplasmid-bornerrnBoperonwithoutthehGHgene.SpecificityandEfficiencyoftheSpecializedRib
45、osomeSys-tem.ToinvestigatetherelativeefficiencyandmRNAspec-ificityofthespecializedribosomesintranslatingendogenouswild-typemessengers,itisessentialtoinactivatethewild-typeribosomesderivedfromchromosomalrrnoperons.Hence,thespectinomycin-resistancemutation(18)wasin-troducedintotherRNAofthespecializedr
46、ibosomes.Thespecializedribosomesweremadespectinomycin-resistantbyintroducingaC-Usubstitutionatposition1192ofthe16SrRNAmoleculebysite-directedmutagenesis(10).There-sultingplasmidspASDVIII(IXandX)-PSDRVIII(IXandX)-PL-SpCrwereusedtotransformE.coliK5637,whichcontainsthetemperature-sensitiveXrepressor.Af
47、ter2hrofinduction,spectinomycinwasaddedtoinactivatethewild-typeribosomes;5minlater,35Smethioninewasaddedtolabeltheproteinstranslatedbythespectinomycin-resistantspecializedribosomes.TheradioactiveproteinprofilewasanalyzedbyNaDodSO4/polyacrylamidegelelectrophoresisfollowedbyautoradiography(Fig.5).Thep
48、roteinprofileofcellscontainingsystemVIII-Spcr(lane1)is,asexpected,similartothatofcellsgrownintheabsenceoftheantibiotic(lane10).IntheproteinprofilesofcellshavingthespecializedsystemsIX-SpcrandX-Spcr(lanes2and3,respectively),hGHisthemostprominentband(25-30%oftotalnewlysynthesizedproteins).Comparisonof
49、thetotalamountoflabeledhGHproducedinbothsystemsconfirmspreviousresultsshowingthattheoverallefficiencyofsystemXis2-to3-foldhigherthanthatofsystemIX.Lanes6-8showthe12345678910amFIG.5.35SMethionineincorporationintoproteinssynthesizedbyspectinomycin-resistantspecializedribosomesinthepresenceofspectinomy
50、cin.ProteinprofilesfromE.ccliK5637(cI857)cellscontainingthevariousplasmidsareshown.Cellsweregrownat30Cfor2hrinM9mediumwithoutCasaminoacids.After2hrofinductionat370C,spectinomycin(GIBCO)wasadded(0.5mg/ml);5minlater,35Slmethionine(760g.Ci/.LM;1Ci=37GBq)wasadded.Proteinswereextractedafter30mmnofincubat