Extending the spacing between the Shine-Dalgarno sequence ...

By forming basepairing interactions with the 3' end of 16S rRNA, mRNA Shine-Dalgarno (SD) sequences positioned upstream of Open Reading Frames ( ... Skiptomaincontent NewResults ExtendingthespacingbetweentheShine-DalgarnosequenceandP-sitecodonreducestherateofmRNAtranslocation HironaoWakabayashi,ChandaniWarnasooriya,ViewORCIDProfileDmitriN.Ermolenko doi:https://doi.org/10.1101/2020.04.16.045807 HironaoWakabayashiDepartmentofBiochemistry&BiophysicsatSchoolofMedicineandDentistryandCenterforRNABiology,UniversityofRochester,Rochester,NY,USAFindthisauthoronGoogleScholarFindthisauthoronPubMedSearchforthisauthoronthissiteChandaniWarnasooriyaDepartmentofBiochemistry&BiophysicsatSchoolofMedicineandDentistryandCenterforRNABiology,UniversityofRochester,Rochester,NY,USAFindthisauthoronGoogleScholarFindthisauthoronPubMedSearchforthisauthoronthissiteDmitriN.ErmolenkoDepartmentofBiochemistry&BiophysicsatSchoolofMedicineandDentistryandCenterforRNABiology,UniversityofRochester,Rochester,NY,USAFindthisauthoronGoogleScholarFindthisauthoronPubMedSearchforthisauthoronthissiteORCIDrecordforDmitriN.ErmolenkoForcorrespondence: [email protected] 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AbstractByformingbasepairinginteractionswiththe3’endof16SrRNA,mRNAShine-Dalgarno(SD)sequencespositionedupstreamofOpenReadingFrames(ORFs)facilitatetranslationinitiation.Duringtheelongationphaseofproteinsynthesis,intragenicSD-likesequencesstimulateribosomeframeshiftingandmayalsoslowdownribosomemovementalongmRNA.Here,weshowthatthelengthofthespacerbetweentheSDsequenceandP-sitecodonstronglyaffectstherateofribosometranslocation.IncreasingthespacerlengthbeyondsixnucleotidesdestabilizesmRNA-ribosomeinteractionsandresultsina5-10foldreductionofthetranslocationrate.Theseobservationssuggestthatduringtranslation,thespacerbetweentheSDsequenceandP-sitecodonundergoesstructuralrearrangements,whichslowdownmRNAtranslocationandpromotemRNAframeshifting.IntroductionTheShine-Dalgarno(SD)sequenceplaysseveralimportantrolesinregulationofproteinsynthesisinbacteria.3-to-9nucleotide-longSDsequences(UAAGGAGGU)occurupstreamofthestartcodonofmostbacterialORFsandincreaseefficiencyoftranslationinitiationthroughbasepairinginteractionswiththeanti-SD(aSD)sequenceatthe3’endof16SrRNA[1,2].SD-likesequences,suchasArg(AGGAGG)orGly(GGAGGU)codonpairs,alsooccurinsideopenreadingframes(ORFs)wheretheymayregulatetranslationelongation.Ribosomeprofilingstudies[3,4]andinvitrosingle-moleculeexperiments[5,6]suggestedthatSD-likesequencespresentwithinORFsofmRNAinduceribosomepausing.SpecificinternalSD-likesequenceswithinORFsareconservedbetweendivergentbacterialspecies[3]indicatingthatSD-inducedpausesmaybefunctionallyimportant.SD-inducedribosomepausingwassuggestedtopreventtheformationofanti-terminationRNAstem-loopsand,thus,enablepropertranscriptiontermination[3].Inaddition,clusteringofSD-likesequencesinmRNAsegmentscodingforloopsinproteinstructureswerehypothesizedtomodulateco-translationalproteinfolding[3].However,morerecentribosomeprofiling[7]andbiochemicalstudies[8]showedthatintragenicSD-likesequencehavelittleornoeffectontherateofribosometranslocationalongmRNA.WhileitremainsunclearwhetherSD-likesequenceinduceribosomepausing,involvementofSDsequencesinprogrammedribosomeframeshifting(PRF)hasbeenwelldocumented[9-11].Forexample,aSDsequenceinduces+1PRF,whichproducesreleasefactor2(RF2)inE.coli.SDsequencesalsostimulate-1PRFeventsthatproduceγsubunitofDNApolymeraseIIIandcytidinedeaminase[11-14].ElucidationoftheeffectofSDsequencesonelongationmaybecomplicatedbythefactthatthestrengthoftheSD-aSDinteractiondiffersfrommRNAtomRNAasthenumberofSD-aSDbasepairsvariesfrom3to9nucleotides.Furthermore,thespacerbetweentheSDsequenceandP-sitecodonwasshowntohaveastrongeffectontheefficiencyofbothtranslationinitiationandSD-stimulatedframeshiftinginbacteria[9,10,15,16].Forexample,SDsequencespositionedlessthan4nucleotidesupstreamofthePsitecodondestabilizeP-sitebindingoftRNAandpromote+1PRF[9,16].ThemostoptimalspacingbetweenSDandtheAofAUGstartcodonforthepromotionoftranslationinitiationis4-9nucleotides[15].Bycontrast,themostoptimalspacingbetweenthePsitecodonandSDsequencesforstimulationof-1PRFindnaXmRNAis10-14nucleotides[10].ThestimulatoryeffectoftheSDsequenceon-1PRFindnaXmRNAdisappearswhenthespacerbetweenSDandtheslipperysequenceisincreasedfrom10to16nucleotidesordecreasedfrom10to7nucleotides[10].ItisnotclearwhythelengthofthespacerbetweentheSDsequenceandP-sitecodonoptimalforinitiationissignificantlydifferentfromthespacingoptimalforthe-1PRF.Ribosomeprofilingdata[4]andindirectbiochemicalexperiments[17]suggestthatSD-aSDinteractionscanbepreservedforanumberofroundsofribosometranslocationthatpossiblyresultsinloopingorinchwormmovementofthespacersequencebetweentheSDandP-sitecodoninsidetheribosome.AtthepointofSD-aSDhybriddissociation,thehybridmaycreatetensionthatpullstheribosomebackandinducesribosomepausingandframeshifting[11].ItislikelythattheeffectoftheSDsequenceontranslocationdependsonthenumberofSD-aSDbasepairsandthespacingbetweentheSDandtranslocatingcodonsinmRNA.Thus,variationsintheseparametersmayunderliethediscrepanciesbetweendifferentstudiesoftheSDinhibitoryeffectonribosometranslocation.Herewe(i)determinetowhatextenttheSD-anti-SDinteractionslowsdownribosometranslocationand(ii)elucidatethedependenceoftranslocationrateonstabilityoftheSD-anti-SDduplexandthespacingbetweentheSDsequenceandP-sitecodon.OurresultsareconsistentwiththeideathatSD-aSDinteractionsareretainedforseveralroundsofribosometranslocationalongmRNA.WealsofindthattheeffectofSD-aSDinteractionsontranslocationishighlyvariableanddependsonthespacingbetweentheSDsequenceandP-sitecodon.OurresultsreconcilepreviouscontradictoryreportsregardingtheroleofSD-likesequencesinregulatingtherateoftranslationelongationandprovidenewinsightsintomechanismsofribosomepausingandframeshifting.ResultsLengtheningthespacerbetweentheSDandP-sitecodondestabilizesmRNA-ribosomeinteractionsTotesthowthelengthofthespacerbetweentheSDsequenceandPsitecodonaffectstabilityofribosome-mRNAinteractionsandribosometranslocation,wemadeeightmodelmRNAsbasedonthederivativeofthephageT4gene32mRNAnamedm301[18,19].IntheseeightmRNA,wevariedspacingbetweenaAAGGASDsequenceandaUAC(Tyr)codonfrom6to21nucleotides.Wefirstexaminedaninitiation-likecomplexcontainingatRNAinthePsite.WeassembledribosomalcomplexeswitheachmRNAvariantandadeacylatedtRNATyrandmeasuredstabilityofribosome-mRNAinteractionsusingtoeprinting.Intoeprintingassay,thepositionoftheribosomealongmRNAismappedbyinhibitionofreversetranscriptasethatextendsDNAprimerannealeddownstreamoftheribosome.TheribosomecontainingP-sitetRNAproducesatoeprint16nucleotidesdownstreamfromthefirstnucleotideofthePsitecodon.ThepresenceofSD-aSDinteractionsstabilizesthemRNA-ribosomecomplexandsubstantiallyincreasesintensityofthetoeprint[16,18].ComparisonoftoeprintintensitiesbetweenmRNAswithdifferentSD-UACspacingindicatedthatstabilityofmRNA-ribosomecomplexprogressivelydecreasedasspacingbetweentheSDsequenceandP-sitecodonwasextendedfrom6to11nucleotides(Fig.1).ToeprintsbecamebarelydetectablewhenspacingbetweentheSDandP-sitecodonwasextendedbeyond11nucleotides.TheseresultssuggestthatelongatingspacingbetweentheSDsequenceandPsitecodonbeyondthelengthwhichwasshowntobeoptimalforstimulatingtranslationinitiation,destabilizesmRNA-ribosomeinteractions.DownloadfigureOpeninnewtabFigure1.ExtendingspacingbetweentheSDsequenceandthePsitecodondestabilizesmRNA-ribosomeinteractions.Toeprintswereproducedbyincubatingthe70SribosomewithanmRNAandtRNATyr,whichbasepairswithauniqueUACcodondownstreamoftheSD.EachmRNAcontainedeitherAAGGA,AAGGAGGUorUAAaGAGGUSDsequenceasindicated.ThespacerbetweentheSDandUACcodonvariedbetween6and21nucleotidesinlengthasindicated.WenextaskedhowthespacerlengthaffectsmRNA-ribosomeinteractionsinanelongationcomplex,whichcontainstwotRNAs.BindingofanA-sitetRNAstabilizedmRNA-ribosomesinteractions.IncontrasttoribosomecomplexescontainingasingleP-sitetRNA(Fig.1),pre-translocationelongationcomplexescontainingbothA-andP-sitetRNAsproduceddetectabletoeprintsevenwhenspacingbetweentheSDsequenceandUACcodonwasaslongas21nucleotides(Suppl.Fig.1).Nevertheless,thedependenceonthespacerlengthfollowedthesametrendasforasingletRNA,initiation-likecomplex.ThestabilityofmRNA-ribosomecomplexprogressivelydecreasedasthespacingbetweentheSDsequenceandP-sitecodonincreasedfrom6to21nucleotides(Suppl.Fig.1).Wenexttestedwhetherthedependenceofribosome-mRNAinteractionsonspacingbetweentheSDandP-sitecodonisalteredwhenthestrengthofSD-aSDinteractionsisincreased.Tothatend,wereplacedtheAAGGASDsequenceinalleightoriginalmRNAvariantswithAAGGAGGUthatispredictedtoincreasethethermodynamicstabilityofSD-aSDduplexfrom-3.6kcal/molto-11.2kcal/mol.IncontrasttomRNAscontainingAAGGASDsequence,extendingspacingbetweenAAGGAGGUSDsequenceandUACcodonfrom6to9nucleotidesincreasedintensityofthetoeprintproducedbytheribosomecontainingP-sitetRNATyr.FurtherextensionofthespacerbetweentheSDandUACcodongraduallydecreasedthetoeprintintensity,yetthetoeprintremainreadilydetectableuntilthespacingwasextendedto17nucleotides.Thus,AAGGAGGUSDsequenceincreasedstabilityoftheribosome-mRNAcomplexrelativetoAAGGASDsequence.Inaddition,SDsequencesAAGGAandAAGGAGGUdifferinoptimalspacingbetweentheSDandP-sitecodon,atwhichmRNA-ribosomeinteractionaremoststable(6and9nucleotides,respectively).WefurtherexaminedwhetherthesedifferencesinoptimalSD-P-sitespacingatwhichmRNA-ribosomeinteractionaremoststable,areduetodifferencesinstrengthofSD-aSDinteractionsorduetodistinctalignmentsofSDsequenceswiththeaSDsequenceof16SrRNAthatcouldaffectSD-P-sitespacing.TheAAGGASDsequenceannealsto16SrRNAnearthe3’endofaSDsequence(nt1537-1541inE.coli)whileAAGGAGGUSDsequencespanstheentireaSDsequenceincludingits5’end(nt1534-1541inE.coli).TodestabilizeSD-aSDinteractionsforthelongSDsequence,weintroducedonemismatchinSD-aSDduplexbyreplacingAAGGAGGUSDwithUAAaGAGGUSD(lowercaseletter“a”indicatesmismatchedadenine)thatispredictedtochangethermodynamicstabilityofSD-aSDduplexfrom-11.2kcal/molto-6.4kcal/mol.AmismatchinSD-aSDduplexdecreasedintensityoftoeprintsrelativetotoeprintsobservedinthepresenceofmRNAscontainingAAGGAGGUSD,indicatinglowerstabilityofmRNA-ribosomecomplexinribosomesprogrammedbymRNAswithUAAaGAGGUSD.However,similartomRNAswithAAGGAGGUSD,theoptimalspacingbetweenUAAaGAGGUSDandP-sitecodon,atwhichmRNA-ribosomeinteractionsaremoststable,was9nucleotides.Hence,optimalSD-P-sitespacingatwhichmRNA-ribosomeinteractionaremoststableisatleastpartiallydefinedbythealignmentofSDsequencesrelativetoaSDsequence.Takentogether,toeprintingexperimentsindicatethateachSDsequencehasaspecificoptimumforspacingbetweentheSDandP-sitecodon,atwhichmRNA-ribosomeinteractionsaremoststable.ExtendingthespacerbetweentheSDandP-sitecodonbeyondthisoptimallengthdestabilizesmRNA-ribosomeinteractions.TheseresultssuggestthatSD-aSDinteractionsmaybemaintainedforseveralroundsoftranslationelongationresultingininchworm-likemovementofthespacerbetweentheSDsequenceandP-sitecodon.However,thisrearrangementofthespacerdestabilizesmRNA-ribosomeinteractions.ExtendingthespacingbetweentheSDandP-sitecodonpromotesmRNAback-slippageInsideORFs,SDsequencesareknowntopromotePRF.WeusedourmodelmRNAstoexaminehowspacingbetweentheSDandP-sitecodonaffectframemaintenance.IthasbeenpreviouslydemonstratedthattranslocationofdeacylatedtRNA(butnotpeptidyl-tRNA)fromtheAtoPsiteoftheribosomecanbeaccompaniedbyarepairingofthetRNAanticodonwiththeupstreamsynonymouscodon,whichispositionedclosertotheSDsequence(Fig.2)[18,20].ThiseffecthasbeencalledmRNAback-slippage.SuchmRNAback-slippageisthoughttobedrivenbytheSDsequencepositionedatamorefavorabledistancefromtheupstreamcodon[18,20].Themechanismofthisphenomenon,whichisobservedinvitro,islikelysimilartothemechanismofSD-driven-1PRFoccurringinvivo.DownloadfigureOpeninnewtabFigure2.ExtendingspacingbetweentheSDandP-sitecodonpromotesmRNAback-slippage.Apre-translocationcomplex(“Pre-transl”)wasmadebybindingdeacylatedtRNATyrtothePsiteanddeacylatedtRNAPhetotheAsiteinthepresenceofanmRNA.EachmRNAcontainedeitherAAGGA,AAGGAGGUorUAAaGAGGUSDsequenceasindicated.ThespacerbetweentheSDandPhe2UUUcodonwaseither9,12or14nucleotidesasindicated.ThepositionoftheribosomealongthemRNAwasmappedbytoeprinting.Pre-translocationcomplexeswereincubatedwithEF-GandGTPtoinducemRNAtranslocation(+Glanes).“Post-transl”toeprintbandscorrespondtotheproductofaccuratetranslocation,inwhichP-sitetRNAPheisbasepairedwithPhe2UUUcodon.“Slip”bandscorrespondtotheproductofmRNAback-slippage,inwhichP-sitetRNAPheisbasepairedwithPhe1UUUcodon.Percentofback-slippagewascalculatedfromtheintensityof“slip”bandnormalizedtothesumofintensitiesof“slip”and“post-transl”bands.WeusedourmodelmRNAstoexaminehowthespacingbetweentheSDandP-sitecodonaffectsframemaintenance.mRNAback-slippagewaspreviouslyobservedinmRNA301[19,21],fromwhichwederivedaforementionedmodelmRNAs.InthesemodelmRNAs,downstreamofSDsequence,therearetwoalternativecodonsfortRNAPheflankingtheUAC(Tyr)codon(Fig.2).TotestwhetherefficiencyofmRNAback-slippagedependsonthespacingbetweenSDsequenceandthetwoPhecodons,toeprintingexperimentswereperformed.DeacylatedtRNATyrwasfirstboundtothePsiteoftheribosomeprogrammedwithoneofourmodelmRNAs.Next,deacylatedtRNAPhewasboundtotheAsitetobasepairwiththedownstream(Phe2)codon.Theformationofpre-translocationcomplexresultsintheappearanceofthedoublettoeprintcharacteristicoftheribosomecontaininganA-sitetRNA(Fig.2).TranslocationinducedbyEF-GandGTPresultedintheappearanceofatoeprintthatcorrespondstothepost-translocationribosomecontainingP-sitetRNAPhebasepairedwiththedownstreamPhecodon(“post-transl”,Fig.2).mRNAback-slippageproducedadditionaltoeprintthatcorrespondstotheribosomecontainingP-sitetRNAPhebasepairedwiththeupstream(Phe1)codon(“slip”,Fig.2).EfficiencyofmRNAback-slippagewasnegligible(≤10%ofribosomes)whenthespacingbetweentheAAGGASDandthedownstreamPhecodon(Phe2)was9nucleotides.However,extendingspacingbetweenSDandthedownstreamPhecodon(Phe2)to12or14nucleotidesincreasedback-slippageefficiencyto70%(Fig.2).OurtoeprintingexperimentsshowthatextendingthespacingbetweentheSDandthedownstreamPhecodon(Phe2)destabilizescodon-anticodoninteractionsandstimulatesre-pairingofP-sitetRNAPhewithanupstreamPhecodon(Phe1),whichispositionedfivenucleotidesclosertotheSDsequence.ThestrongerAAGGAGGUsequencewiththeSD-Phe2spacingof9nucleotideshasasubstantiallyhigherback-slippageefficiency(60%)thanthatofAAGGA(Fig.2).IntroducingamismatchintotheSD-aSDhelixbychangingAAGGAGGUtoUAAaGAGGUdecreasedback-slippageefficiencyfrom60%(withAAGGAGGUSD)to30%(withUAAaGAGGUSD)(Fig.2).SimilarlytoAAGGA,theUAAaGAGGUsequencewiththeSD-Phe2spacingextendedto12-14nucleotidesincreasesefficiencyofmRNAback-slippagefrom30to50-60%(Fig.2).OurtoeprintingexperimentssuggestthatthemechanismofSD-stimulated-1PRFinbacteriainvolvesSD-drivendestabilizationofcodon-anticodoninteractions,whichleadstomRNAback-slippage.ThisSD-drivendestabilizationofcodon-anticodoninteractionsdependsonspacingbetweenSDandP-sitecodon.OurtoeprintingresultsareconsistentwithexperimentsdemonstratingthatshorteningspacingbetweenP-sitecodonandtheSDsequencefrom10to7nucleotidesinhibits-1PRFindnaXmRNA[10].ExtendingthespacingbetweentheSDandP-sitecodoninhibitsribosomeintersubunitrotationcoupledtomRNAtranslocationWenextexaminedhowthestrengthofSDsequenceandspacingbetweenSDandP-sitecodonaffecttherateofribosometranslocation.Ribosometranslocationiscoupledtocyclicrotationalmovementbetweenribosomalsubunits[22,23].Uponformationofanewpeptidebond,ribosomalsubunitsrotaterelativetoeachotherbyupto10°fromanonrotatedconformationintoarotatedconformation[22,23].BindingofEF-Gtransientlystabilizestherotatedconformation,andcatalyzesmRNAtranslocationduringthereverserotationbetweensubunits[22,23].Försterresonanceenergytransfer(FRET)betweenfluorophoresattachedtoribosomalproteinsS6andL9waspreviouslyextensivelyusedtofollowintersubunitrotation[24-28].ThereverseintersubunitrotationfromrotatedtononrotatedconformationincreasesFRETbetweenfluorophoresattachedtoproteinsS6andL9.Here,weusedtheFRETassaytoexaminehowSDsequencesandSD-P-sitespacingaffecttranslocation.Tothisend,wemeasuredpre-steady-statekineticsofthereverserotationbetweensubunitsoftheribosomesprogrammedwithaforementionedmodelmRNAs,whichdifferinSDsequencesandspacingbetweenP-sitecodonandSD.Toassemblepre-translocationribosomes,deacylatedtRNATyrwasfirstboundtothePsiteoftheribosomeprogrammedwithamodelmRNA.Next,N-acetyl-Phe-tRNAPhewasboundtotheAsite.Consistentwithpublishedreports[18,20],nomRNAback-slippagewasobservedwhenpeptidyl-tRNA(N-acetyl-Phe-tRNAPhe)translocatedfromtheAtoPsiteuponadditionofEF-GandGTP(Suppl.Fig.1).Whenpre-translocationribosomesweremixedwithEF-GandGTPinastopped-flowfluorometer,weobservedarapidincreaseinacceptorfluorescenceindicatingincreaseinFRET(Fig.3a).TheFRETincreasecorrespondstothereverserotationofribosomalsubunitsintonon-rotatedconformationoftheribosomethataccompaniesmRNAtranslocation[26].Kinetictraceswerebestfittedtoadouble-exponentialfunction.Bi-phasickineticsofribosometranslocationwaspreviouslyobservedinmultiplestudies[26,29-31].Itisnotfullyknownwhetherthebi-phasickineticsoftranslocationisduetoheterogeneityofribosomepopulationorotherfactors.Thefasterrateconstantk1,whichaccountsfor55-70%oftheamplitudeoffluorescencechange,istypicallyusedasameasureoftranslocationrate.DownloadfigureOpeninnewtabFigure3.KineticsofintersubunitrotationcoupledtotranslocationofmRNAswithdifferentspacingbetweentheSDandP-sitecodon(UAC).Pre-translocationS6-Alexa488(donor)/L9-Alexa568(acceptor)ribosomescontainingdeacylatedtRNATyrinthePsiteandN-acetyl-Phe-tRNAPheintheAsitewererapidlymixedwithEF-GandGTP.Pre-translocationribosomeswereprogrammedbymRNAscontainingaAAGGASDsequence.SpacingbetweentheSDandP-site(UAC)codonvariedfrom6to21nucleotidesasindicated.(a)KineticsofintersubunitrotationfollowedbyFRET(acceptorfluorescence)inribosomesprogrammedbymRNAwith6-nucleotidelongSD-UACspacer(blue);mRNAwith21-nucleotidelongSD-UACspacer(cyan);mRNAwith21-nucleotidelongSD-UACspacerannealedtoeitherananti-SDDNAoligo(red)oracontrolDNAoligo(magenta).Double-exponentialfitsareblacklines.(b)Bargraphsshowingratesofintersubunitrotation(k1ofdouble-exponentialfit)coupledtotranslocationofmRNAswithvariousSD-UACcodonspacing(blue).RatesmeasuredinribosomesprogrammedwithmRNAsannealedtoeitherananti-SDDNAoligooracontrolDNAoligoareshowninredandmagenta,respectively.RibosomesprogrammedbymRNAwithan6nucleotide-longspacerbetweentheAAGGASDsequenceandP-site(UAC)codontranslocatedrapidly(k1=7.2±0.8s-1).ExtendingspacingbetweentheAAGGASDsequenceandUACcodonto9nucleotidessubstantiallyslowedtherateofreverseintersubunitrotationcoupledtomRNAtranslocationto1.5s-1(Fig.3b).Evenlowerratesof0.6-0.8s-1wereobservedformRNAswitha11,13,15,17,19or21-nucleotidelongspacerbetweentheAAGGASDsequenceandP-site(UAC)codon(Fig3b,Suppl.Table1).ExtendingtheSD-P-sitespaceralsodecreasedtheamplitudeoffluorescencechange(Fig.3a)likelyindicatinglowerstabilityofpre-translocationcomplexesprogrammedbythesemRNAs(Fig.1andSuppl.Fig.1).TheseresultssuggestthatextendingthespacingbetweentheSDsequenceandP-sitecodoninhibitstheintersubunitrotationcoupledtomRNAtranslocation.ApossiblealternativeinterpretationofthekineticsexperimentsisthatextendingthespacingbetweentheAAGGASDsequenceandUACcodonbeyond9nucleotidesdisruptsSD-aSDinteractions.TomeasuretherateoftranslocationintheabsenceofSD-aSDinteractions,DNAoligocomplementarytoSDsequenceand15nucleotidesupstreamofSDwasaddedtopre-translocationribosomesprogrammedwithanmRNA,whichcontainedeither19or21nucleotide-longSD-P-sitespacer.Anti-SDDNAoligoisexpectedtocompetewithaSDsequenceof16SrRNAandthussequesterSDsequenceintoDNA-RNAduplex.Annealinganti-SDDNAoligodramaticallyincreasedtherateofribosometranslocationfrom0.6to4.5s-1for19-nucleotidespacing,andfrom0.6to7.5s-1for21-nucleotidespacingbetweenSDandUACcodon(Fig.3,Suppl.Table1).Asanegativecontrol,insteadofanti-SDDNAoligo,20-nuleotidelongDNAoligocomplementarytomRNAsequenceupstreamofSD(butnotSDsequenceitself)wasaddedtopre-translocationcomplexeswith19or21spacersbetweenSDandUACcodon.Incontrasttoanti-SDDNAoligo,annealingcontrolDNAoligosdidnotincreasetherateoftranslocation(Fig.3andSuppl.Table1).OurresultssuggestthatSD-aSDinteractionsremainintactandslowdowntranslocationevenwhenthespacerbetweenSDandUACcodonis19or21-nucleotideslong.Remarkably,ribosomesprogrammedbymRNAwith6-nucleotidelongspacerbetweenSDandUACcodonandribosomesprogrammedbymRNAwith21-nucleotidelongspacer,inwhichSD-aSDinteractionsweredisruptedbytheanti-SDDNAoligo,translocatedessentiallyatthesamerate.Hence,whenspacingbetweenSDandP-sitecodonisshort,mRNAtranslocationisequallyfastastranslocationintheabsenceofSD-aSDinteractions.However,extendingtheSD-PsitespacerslowsdowntranslocationuntilSD-aSDhelixisunwound.Inthecontextofa6-nucleotidespacerbetweenSDandUAC(P-site)codon,replacingtheAAGGASDsequencewiththestrongerSDsequenceAAGGAGGU,decreasedtherateoftranslocationfrom7.2(AAGGASD)to1.5s-1(AAGGAGGUSD)(Fig.4,Suppl.Table2).InthepresenceofthestrongerSDsequenceAAGGAGGU,extendingSD-P-sitespacerfrom6to9or11nucleotidesdecreasedtherateoftranslocationfrom1.5to0.6or0.8s-1,respectively(Fig.4,Suppl.Table2).Hence,increasingthestrengthofSD-aSDinteractionsslowsdownribosometranslocation.DownloadfigureOpeninnewtabFigure4.KineticsofintersubunitrotationcoupledtotranslocationofmRNAscontainingdifferentSDsequences.Pre-translocationS6-Alexa488(donor)/L9-Alexa568(acceptor)ribosomescontainingdeacylatedtRNATyrinthePsiteandN-acetyl-Phe-tRNAPheintheAsitewererapidlymixedwithEF-GandGTP.Pre-translocationribosomeswereprogrammedbymRNAscontainingeitherAAGGA(blue),AAGGAGGU(black)orUAAaGAGGU(green)SDsequence.SpacingbetweentheSDandP-site(UAC)codonvariedfrom6to11nucleotidesasindicated.Consistentwiththisconclusion,introductionofamismatchintoSD-aSDduplexinthecontextof6-nucleotidespacerbetweenSDandUAC(P-site)codon,increasedtherateoftranslocationfrom1.5(withAAGGAGGUSD)to3.5s-1(withUAAaGAGGUSD)(Fig.4,Suppl.Table3).InthepresenceofUAAaGAGGUSDsequence,extendingSD-P-sitespacerfrom6to9or11nucleotidesdecreasedtherateoftranslocationfrom3.5to0.9or1.1s-1,respectively(Fig.4,Suppl.Table3).OurkineticdataindicatethatregardlessofSDsequence,extendingthespacingbetweentheSDandP-sitecodonslowsdowntranslocation.However,inthepresenceofthestrongerSDsequence,variationinSD-P-sitespacerlengthhasasmallereffectontranslocationrate.IncontrasttoAAGGASDsequence,thestrongerSDsequenceAAGGAGGUsubstantiallyinhibitstranslocationevenwhenthespacerbetweenSDandP-sitecodonisasshortas6nucleotides.ExtendingthespacingbetweentheSDandP-sitecodoninhibitsmRNAtranslocationApossiblecaveatinaforementionedkineticexperimentsisthatwemeasuredtherateofmRNAtranslocationindirectlybymeasuringtherateofreverseintersubunitrotationoftheribosomefromtherotatedintonon-rotatedconformation,whichhasbeenpreviouslyshowntobecoupledtomRNAtranslocation[26].TodeterminetherateofmRNAtranslocationdirectly,wemeasuredfluorescencequenchingofafluoresceindyeattachedtothe3’endofshortmodelmRNAsastheymovewithintheribosome.Thisfluorescencequenchingtranslocationassaywasextensivelyusedbefore[26,32-34].WetestedtheeffectofthelengthofthespacerbetweentheSDsequenceandP-site(AUG)codonontherateoftranslocationinthepresenceofastrong(AAGGAGGU)SDsequence.WealsomeasuredtherateoftranslocationofaleaderlessmRNA(5’AUGUACAAAGUAUAA3’)thatdoesnotcontainaSDsequence.Whenpre-translocationribosomes,whichwereassembledwithfluorescein-labeledmRNA,deacylatedtRNAMetinthePsiteandN-acetyl-Tyr-tRNATyrintheAsite,weremixedwithEF-G•GTPusingastopped-flowapparatus,rapidquenchingoffluoresceinfluorescencewasobserved,indicativeofmRNAtranslocation(Fig.5).Consistentwithpublishedreports[26,29-31]andourS6Alexa488/L9Alexa586kineticFRETexperiments(Fig.3),kinetictracesofmRNAtranslocationwerebestfittedtoadouble-exponentialfunction.DownloadfigureOpeninnewtabFigure5.KineticsofmRNAtranslocationmeasuredbyquenchingoffluoresceinattachedtothe3’endofmRNA.Pre-translationribosomescontainingdeacylatedtRNATyrinthePsiteandN-acetyl-Phe-tRNAPheintheAsitewererapidlymixedwithEF-GandGTP.Pre-translocationribosomeswereprogrammedbyfluorescein-labeledmRNAs.AllmRNAscontainedaAAGGAGGUSDsequenceexceptforthe“noSD”leaderlessmRNA,whichlackedtheSD.SpacingbetweentheSDandP-site(UAC)codonvariedfrom4to14nucleotidesasindicated.(a)KineticsoffluoresceinquenchingcorrespondingtotranslocationofmRNAwith4nucleotide-longspacingbetweentheSDandUACcodon(blue),mRNAwith10nucleotide-longspacingbetweentheSDandUACcodon(red)andleaderlessmRNAlackingtheSD(magenta).Double-exponentialfitsareblacklines.(b)RatesofmRNAtranslocation(k1ofdouble-exponentialfit).SimilarlyfasttranslocationwasobservedforthemRNAswiththe4and6nucleotidespacerbetweentheSDandP-sitecodonAUGasfasterrateconstantsk1ofthebi-phasicfitwere4.6and4.1s-1,respectively(Fig.5andSuppl.Table4).Bycontrast,2.5-3folddecreasesintranslocationrateswereobservedformRNAshavinga7,10or14-nucleotidespacer(Fig.5,Suppl.Table4).AmplitudeoffluoresceinquenchingobservedforleaderlessmRNAthatlackstheSDsequencewassignificantlylowerthantheamplitudeobservedformRNAscontainingtheSD(Fig.5).ThisdecreaseinamplitudeislikelyduetomarkedlylowerstabilityofmRNA-ribosomecomplexintheabsenceoftheSD.Nevertheless,leaderlessmRNAtranslocatedslightlyfaster(withtherateconstantk1of7.1s-1)thanmRNAswith4and6-nucleotidelongspacersbetweentheSDandP-sitecodon(Fig.5andSuppl.Table4).Takentogether,ourkineticsmeasurementsshowthatwhenthespacerbetweentheSDandPsitecodonextendsbeyond4-6nucleotides,theSD-aSDhelixappreciablyslowsdowntranslocation.DiscussionOurexperimentsrevealedthattheeffectofSDsequenceonribosometranslocationisvariableanddependsonspacingbetweentheSDandP-sitecodon.Inmostofourexperiments(exceptthemRNAderivedfromm301mRNA,whichcontainedstrongSDsequenceAAGGAGGU),whentheSD-P-sitespacerwas4-6nucleotideslong,therateoftranslocationwassimilartotherateobservedintheabsenceofSD-aSDinteractions(Fig.3and5).ThisresultisconsistentwithexperimentsperformedbyBorgandEhrenbergshowingthatthreedifferentSD-likesequencespositionedsixnucleotidesupstreamofP-sitecodonhadnoeffectontranslocationrate[8].OurdataalsoindicatethatextendingspacingbetweentheSDsequenceandP-sitecodonbeyond6nucleotidessubstantiallyinhibitsribosometranslocation(Fig.3-5).SuchSD-aSDinteractionscanresultin5-10foldreductionofribosometranslocationratewhencomparedtotherateobservedintheabsenceofSD-aSDinteractions.Theseobservationsareconsistentwithpreviousinvitrosingle-moleculeexperimentsdemonstratingthatSDsequencesslowsdowntranslocationby3-4foldwhenthespacerbetweenSDsequenceandP-sitecodonis9-15nucleotideslong[6].Onlyat6nucleotide-longSD-P-sitespacing,theinhibitoryeffectofSD-aSDinteractionsinverselycorrelatedwiththerateoftranslocation:strongestSDsequenceAAGGAGGUsloweddowntranslocationtothelargestdegree(Fig.4).Bycontrast,mRNAsthatcontainedAAGGA,AAGGAGGUorUAAaGAGGUSDsequences7-14nucleotidesupstreamofthePsiteweretranslocatedatsimilarrates(Fig.3-5)suggestingthatatleastunderexperimentalconditionsusedinourinvitrostudies,theeffectofSDsequenceontranslocationrateprimarilydependsontheSD-P-sitespacingandnotthestrengthofSD-aSDinteractions.InmodelmRNAsusedinthisstudy,theSD-P-sitespacerwasextendedbyinsertingintrinsicallyunstructuredCArepeats.Unexploredinourstudy,nucleotidecompositionandbasepairingpotentialoftheSD-P-sitespacersequencemayalsoaffecttherateoftranslocationthusincreasingvariabilityoftheeffectofSDsequenceonribosometranslocation.Ourkineticexperimentsperformedinthepresenceofanti-SDDNAoligoannealedtomRNAcontainingashortSDsequenceAAGGA(Fig.3)indicatethatatleastunderconditionsusedinourinvitroexperiments,SD-aSDinteractionscanberetainedwhenthespacerbetweenSDandPsitecodonis≤21-nucleotidelong.Hence,uponprogressivemovementoftheribosomeawayfromtheSDsequence,mRNAlikelyundergoesinchwormrearrangementinsidetheribosome.ThisideaissupportedbyinvivoribosomeprofilingdatademonstratingthatSD-likesequencesproduceribosome-protectedfootprintsthatareupto15nucleotidelongerthantypical28-29nt-longribosomeprotectedmRNAfragments[4].Furthermore,theselongerribosome-protectedfragmentsareasymmetricallyelongatedatthe3’end,consistentwithinchworm-likerearrangementofmRNAanchoredbySDsequencetothe30Ssubunit.PublishedstructuralstudiesprovidepossiblecluesforthemechanismofSD-inducedinhibitionoftranslocation.X-raycrystalstructuresofseveralribosome-mRNAcomplexeswithdifferentSD-P-sitespacingindicatedthatduringthefirstofseveralelongationcycles,SD-aSDhelixundergoesrotationalmovementandshiftsawayfromtheintersubunitinterfacetowardthesolventsideofthe30Ssubunitclosertothe30Sheaddomain[35,36].Inthecrystalstructureoftheribosomewith9nucleotidespacerbetweentheSDandP-sitecodon,threenucleotidesofthespaceradjacenttoSDwerestackedontopofbasepairsformedbySDand16SrRNAthusextendingSD-aSDhelix[36].ExtensionofthespacerbetweenSDandP-sitecodonmayalsoleadtootherstructuralchangesthatareyettobevisualizedbyX-raycrystallographyandcryo-EM.WehypothesizethatmRNArearrangementsresultingfromtheextensionofSD-P-sitespacercauseinhibitionofmRNAtranslocation.Onepossibilityisthatthesestructuralrearrangementsinterferewiththerotationalmovementofthe30SheaddomainthatisthoughttoaccompanymRNAtranslocation[37].OurworkprovidesnewinsightsintotherolesofSDsequencesintranslationinitiationandelongation.TheinhibitionoftranslocationbySDsequencesdetectedinourexperimentsmayaffectthetransitionfrominitiationtoelongationphaseofproteinsynthesis[38]andatleastpartiallyberesponsiblefortheslowertranslationofthefirstseveralcodonsinORFsthathasbeenobservedinseveralpreviousstudies[8,39].OurdataalsosupporttheideathatintragenicSDsequencescanregulatetranslationelongation.AlthoughpublishedribosomeprofilingstudiesproducedconflictingdataregardingtheabilityofSD-likesequencestoinducetranslationalpausesinvivo[3,7],SD-aSDinteractionslikelyslowdownribosometranslocationtoacertaindegreeinlivecells.Itispossiblethe5-10foldreductionofribosometranslocationrateobservedinourexperimentsisnotsufficienttoinducelengthytranslationpausesthatcanbedetectedbyribosomeprofilingapproach.ItisalsopossiblethattheeffectofSD-aSDinteractionsonribosometranslocationisexacerbatedunderinvitroconditionsbecauseconcentrationoffreeMg2+ions,whichlikelystabilizeSD-aSDinteractions,islowerinlivecells.Hence,incomparisontoinvitroexperiments,SD-inducedinhibitionoftranslocationmaylessprominentinlivecells.Nevertheless,conservationofintragenicSD-likesequencesbetweendivergentbacterialspecies[2]supporttheideathatSD-mediatedregulationoftranslationelongationisfunctionallyimportantinvivo.Toeprinting(Fig.1-2)andkinetic(Fig.3-5)dataalsoprovideinsightsintothemechanismofSD-stimulatedprogrammedribosomeframeshifting(PRF)inbacteria.OurresultssuggestthatextendingSD-P-sitespacerbeyond9nucleotidesdestabilizesmRNA-ribosomeinteractionsandpromotesmRNAback-slippage.Consistentwiththeseobservations,theoptimalspacingbetweenthePsitecodonandSDsequencesforstimulationof-1PRFinE.colidnaXmRNAwasshowntobe10-14nucleotides[10].Therefore,SDsequenceslikelystimulate-1PRFby(i)slowingdownribosometranslocationand(ii)destabilizingofcodon-anti-codoninteractionsinthe“0”readingframe.MaterialsandMethodsMaterialsandsamplepreparationtRNAPheandtRNATyrwerepurchasedfromChemblock.MRE600E.coli70Sribosomes,6-histidine-taggedEF-GandaminoacylatedtRNAswerepreparedaspreviouslydescribed[18,24].UsingtheQuickChangeSite-DirectedMutagenesisSystem(Agilent,SantaClare,CA),DNAtemplatesencodingmRNAvariantsfortoeprintingandkineticexperimentswerederivedfrompFK301plasmid,whichencodesm301mRNA(5’GUAAAGUGUCAUAGCACCAACUGUUAAUUAAAUUAAAUUAAAAAGGAAAUAAUGUUUACUUUGUAAAAUCUACUGCUGAACUCGCUGCACAAAUGGCUAAACUGAAUGGCAAUAAAGGUUUUUCUUCUGAAGAUAAAG3’;theSDsequenceandaUACcodonareunderlined[19]).SequencesofallmRNAvariantsareshowninSuppl.Table4.Fluorescently-labeledmRNAsandribosomesS6-Alexa488/L9-Alexa568ribosomeswerepreparedaspreviouslydescribed[24,27].SinglecysteinemutantsofthesmallribosomalsubunitproteinS6andlargeribosomalsubunitproteinL9werelabeledusingmaleimidederivativesofAlexa488andAlexa568,respectively(ThermoFisherScientific,Waltham,MA).LabeledS6andL9proteinswereincorporatedintoΔS630SandΔL950Ssubunits,respectively,bypartialreconstitutionaspreviouslydescribed[24,27].FluoresceinlabeledmRNAsweresynthesizedbyIntegratedDNATechnologies(Coralville,IA).mRNAvariantswerederivedfromthemRNAwith4-nucleotidespacerbetweenSDandAUGcodon[33](5’GGCAAGGAGGUAAAAAUGUACAAAGUAUAA3’Fluorescein;SDsequenceandAUGcodonareunderlined):5’GGCAAGGAGGUACACAAAUGUACAAA3’Fluorescein(6ntspacer);5’GGCAAGGAGGUACACAAAAUGUACAAA3’Fluorescein(7ntspacer);5’GGCAAGGAGGUACAACACAAAAUGUACAAA3’Fluorescein(10ntspacer);5’GGCAAGGAGGUAACAACACAAACAAAUGUACAAA3’Fluorescein(14ntspacer).WealsomeasuredtherateoftranslocationofleaderlessmRNA(5’AUGUACAAAGUAUAA3’Fluorescein)thatdoesnotcontainSDsequence.ToeprintingexperimentsToeprintingexperimentswereperformedinpolyaminebuffer(30mMHEPES•KOH,pH7.5,70mMNH4Cl,6mMMgCl2,2mMspermidine,0.1mMspermine)aspreviouslydescribed[18,21].ComplexesinFig.1wereassembledbyincubating70Sribosomes(1μM)withdeacylatedtRNATyr(2μM)andmRNA(2μM)preannealedto[32P]-labeledprimer[18]for15minutesat37°C.Thepre-translocationcomplexesinSuppl.Fig.S1wereassembledbyincubating70Sribosomes(1μM)withdeacylatedtRNATyr(2μM)andmRNA(2μM)preannealedto[32P]-labeledprimer[18]for15minutesat37°C.Then,1.5µMN-acetyl-Phe-tRNAPhewasaddedtoP-sitetRNA-boundcomplexesfollowedbyincubationat37°Cfor20minutes.Thepre-translocationcomplexesinFig.2wereassembledsimilarlyexceptthattheAsitewasfilledwith2µMdeacylatedtRNAPheaddedtoP-sitetRNA-boundcomplexes.Translocationwascarriedoutbytheincubationofpre-translocationcomplexes(0.5μM)with1.5μMEF-Ginthepresenceof0.5mMGTPfor10minutesat37°C.Stopped-flowmeasurementsofpre-steady-statetranslocationkineticsKineticsofribosomeintersubunitrotationcoupledtomRNAtranslocationweremeasuredaspreviouslydescribed[26].Pre-translocationcomplexeswereconstructedbytheincubationofS6-Alexa488/L9-Alexa56870Sribosomes(1.4µM)withmRNA2.8µManddeacylatedtRNATyr(4µM)inpolyaminebuffer(30mMHEPES•KOH,pH7.5,150mMNH4Cl,6mMMgCl2,2mMspermidine,0.1mMspermine,6mMβ-mercaptoethanol)for15minutesat37°C,followedbyanincubationwithN-acetyl-Phe-tRNAPhe(2.8µM)for30minutesat37°C.InexperimentsshowninFig.3,28µManti-SD(5’-TCCTTTTTAATTTAATTTAA-3’)orcontrolDNA(5’-ATTTAATTTAATTAACAGTT-3’)oligoswereannealedtomRNAaftercomplexassemblyfor15minat37°C.Anti-SDandcontrololigoweredesignedtohavesimilaraffinitytomRNA.Pre-translocationribosomesweremixedwithEF-GandGTP(orGTPanalogues)usinganAppliedPhotophysicsstopped-flowfluorometer.Finalconcentrationsaftermixingwere:35nMribosomes,µMEF-G,0.5mMGTPand14µMDNAoligo(ifoligowasaddedtopre-translocationribosomes).Alexa488wasexcitedat490nmandchangesinFRET(Alexa568emission)weredetectedusinga590nmlong-passfilter.Allstopped-flowexperimentsweredoneat23°C;monochromatorslitswereadjustedto9.3nm.IncreasesinFRET(Alexa568fluorescence)inkineticstraceswerebestfittedtothesumoftwoexponentials(y=y0+A1*exp(-k1*t)+A2*exp(-k2*t)),correspondingtotheapparentrateconstantsk1andk2.KineticsofmRNAtranslocationweremeasuredbyfluoresceinquenchingaspreviouslydescribedwithminormodifications[26,32,34].Pre-translocationcomplexeswereconstructedbytheincubationof70Sribosomes(1µM)withfluorescein-labeledmRNA(0.85µM)anddeacylatedtRNAMet(2µM)inpolyaminebuffer(30mMHEPES•KOH,pH7.5,150mMNH4Cl,6mMMgCl2,mMspermidine,0.1mMspermine)for15minutesat37°C,followedbyanincubationwithN-acetyl-Tyr-tRNATyr(1.5µM)for30minutesat37°C.Pre-translocationribosomesweremixedwithEF-GandGTPusinganAppliedPhotophysicsstopped-flowfluorometer(Leatherhead,Surrey,UK).Finalconcentrationsaftermixingwere:35nMribosomes,1µMEF-Gand0.5mMGTP.Fluoresceinwasexcitedat490nmandfluorescenceemissionwasdetectedusinga515nmlong-passfilter.Allstopped-flowexperimentsweredoneat23°C;monochromatorslitswereadjustedto9.3nm.TranslocationofthemRNAresultedinapartialquenchingoffluoresceincoupledtothe3’endofthemRNA(8).TimetraceswereanalyzedusingOrigin(OriginLabCorporation,Northampton,MA).Asreportedpreviously[26,30,31,33],thekineticsofmRNAtranslocationareclearlybiphasicandarebestfittedtothesumoftwoexponentials(y=y0+A1*exp(-k1*t)+A2*exp(-k2*t)),correspondingtotheapparentrateconstantsk1andk2.SupplementaryMaterialsDownloadfigureOpeninnewtabSupplementaryFigure1ExtendingspacingbetweentheSDsequenceandthePsitecodondestabilizesmRNA-ribosomeinteractions.Apre-translocationcomplex(“Pre-transl”)wasmadebybindingdeacylatedtRNATyrtothePsiteandN-acetyl-Phe-tRNAPhetotheAsiteinthepresenceofanmRNA.ThespacerbetweentheAAGGASDsequenceandUAC(P-site)codonvariedbetween6and21nucleotidesinlengthasindicated.ThepositionoftheribosomealongthemRNAwasmappedbytoeprinting.Pre-translocationcomplexeswereincubatedwithEF-GandGTPtoinducemRNAtranslocation(+Glanes).“Post-transl”toeprintbandscorrespondtotheproductoftranslocation.Viewthistable:ViewinlineViewpopupDownloadpowerpointSupplementaryTable1.RatesofintersubunitrotationcoupledtotranslocationofmRNAscontaininganAAGGASDsequencePre-translocationS6-Alexa488(donor)/L9-Alexa568(acceptor)ribosomescontainingdeacylatedtRNATyrinthePsiteandN-Ac-Phe-tRNAPheintheAsitewererapidlymixedwithEF-GandGTP(Fig.3).Pre-translocationribosomeswereprogrammedbymRNAscontainingaAAGGASDsequence.SpacingbetweentheSDandP-site(UAC)codonvariedfrom6to21nucleotidesasindicated.Abouttentraceswereacquiredforeachexperiment.k1andk2areratesconstantsofdouble-exponentialfits.Rateconstantsaveragedfromtwotofourindependentexperimentsareshown.Viewthistable:ViewinlineViewpopupDownloadpowerpointSupplementaryTable2.RatesofintersubunitrotationcoupledtotranslocationofmRNAswithAAGGAGGUSDsequencePre-translocationS6-Alexa488(donor)/L9-Alexa568(acceptor)ribosomescontainingdeacylatedtRNATyrinthePsiteandN-Ac-Phe-tRNAPheintheAsitewererapidlymixedwithEF-GandGTP(Fig.4).Pre-translocationribosomeswereprogrammedbymRNAscontainingaAAGGAGGUSDsequence.SpacingbetweentheSDandP-site(UAC)codonvariedfrom6to21nucleotidesasindicated.Abouttentraceswereacquiredforeachexperiment.k1andk2areratesconstantsofdouble-exponentialfits.Rateconstantsaveragedfromtwotofourindependentexperimentsareshown.Viewthistable:ViewinlineViewpopupDownloadpowerpointSupplementaryTable3.RatesofintersubunitrotationcoupledtotranslocationofmRNAswithUAAaGAGGUSDsequencePre-translocationS6-Alexa488(donor)/L9-Alexa568(acceptor)ribosomescontainingdeacylatedtRNATyrinthePsiteandN-Ac-Phe-tRNAPheintheAsitewererapidlymixedwithEF-GandGTP(Fig.4).Pre-translocationribosomeswereprogrammedbymRNAscontainingaUAAaGAGGUSDsequence.SpacingbetweentheSDandP-site(UAC)codonvariedfrom6to21nucleotidesasindicated.Abouttentraceswereacquiredforeachexperiment.k1andk2areratesconstantsofdouble-exponentialfits.Rateconstantsaveragedfromtwotofourindependentexperimentsareshown.Viewthistable:ViewinlineViewpopupDownloadpowerpointSupplementaryTable4.RatesofmRNAtranslocationdeterminedbyquenchingoffluoresceinattachedtothe3’endofmRNAPre-translationribosomescontainingdeacylatedtRNATyrinthePsiteandN-Ac-Phe-tRNAPheintheAsitewererapidlymixedwithEF-GandGTP.Pre-translocationribosomeswereprogrammedbyfluorescein-labeledmRNAs.AllmRNAscontainedaAAGGAGGUSDsequenceexceptforthe“noSD”leaderlessmRNA,whichlackedtheSD.SpacingbetweentheSDandP-site(UAC)codonvariedfrom4to14nucleotidesasindicated.Abouttentraceswereacquiredforeachexperiment.k1andk2areratesconstantsofdouble-exponentialfits.Rateconstantsaveragedfromtwotofourindependentexperimentsareshown.Viewthistable:ViewinlineViewpopupSupplementaryTable5.SequencesofmRNAsusedintoeprintingandS6-Alexa488/L9-Alexa568kineticexperiments.AcknowledgementsThesestudiesweresupportedbygrantfromtheUSNationalInstituteofHealthR01GM099719(toD.N.E.).WethankAndreiKorostelevforhiscommentsonthemanuscript.AbbreviationsusedSDShine-Dalgarno;aSDanti-Shine-Dalgarno;ORFsopenreadingframes;PRFprogrammedribosomeframeshifting;RF2releasefactor2;FRETFörsterresonanceenergytransfer;EF-GelongationfactorG.References[1].↵SteitzJA,JakesK.HowribosomesselectinitiatorregionsinmRNA:basepairformationbetweenthe3’terminusof16SrRNAandthemRNAduringinitiationofproteinsynthesisinEscherichiacoli.ProcNatlAcadSciUSA.1975;72:4734–8.OpenUrlAbstract/FREEFullText[2].↵SaitoK,GreenR,BuskirkAR.TranslationalinitiationinE.colioccursatthecorrectsitesgenome-wideintheabsenceofmRNA-rRNAbase-pairing.Elife.2020;9.[3].↵LiGW,OhE,WeissmanJS.Theanti-Shine-Dalgarnosequencedrivestranslationalpausingandcodonchoiceinbacteria.Nature.2012;484:538–41.OpenUrlCrossRefPubMedWebofScience[4].↵O’ConnorPB,LiGW,WeissmanJS,AtkinsJF,BaranovPV.rRNA:mRNApairingaltersthelengthandthesymmetryofmRNA-protectedfragmentsinribosomeprofilingexperiments.Bioinformatics.2013;29:1488–91.OpenUrlCrossRefPubMedWebofScience[5].↵WenJD,LancasterL,HodgesC,ZeriAC,YoshimuraSH,NollerHF,etal.Followingtranslationbysingleribosomesonecodonatatime.Nature.2008;452:598–603.OpenUrlCrossRefPubMedWebofScience[6].↵ChenJ,PetrovA,JohanssonM,TsaiA,O’LearySE,PuglisiJD.Dynamicpathwaysof-1translationalframeshifting.Nature.2014;512:328–32.OpenUrlCrossRefPubMedWebofScience[7].↵MohammadF,WoolstenhulmeCJ,GreenR,BuskirkAR.ClarifyingtheTranslationalPausingLandscapeinBacteriabyRibosomeProfiling.CellRep.2016;14:686–94.OpenUrlCrossRefPubMed[8].↵BorgA,EhrenbergM.DeterminantsoftherateofmRNAtranslocationinbacterialproteinsynthesis.JMolBiol.2015;427:1835–47.OpenUrlCrossRefPubMed[9].↵SpanjaardRA,vanDuinJ.TranslationofthesequenceAGG-AGGyields50%ribosomalframeshift.ProcNatlAcadSciUSA.1988;85:7967–71.OpenUrlAbstract/FREEFullText[10].↵LarsenB,WillsNM,GestelandRF,AtkinsJF.rRNA-mRNAbasepairingstimulatesaprogrammed-1ribosomalframeshift.JBacteriol.1994;176:6842–51.OpenUrlAbstract/FREEFullText[11].↵AtkinsJF,BaranovPV,FayetO,HerrAJ,HowardMT,IvanovIP,etal.Overridingstandarddecoding:implicationsofre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Share ExtendingthespacingbetweentheShine-DalgarnosequenceandP-sitecodonreducestherateofmRNAtranslocation HironaoWakabayashi,ChandaniWarnasooriya,DmitriN.Ermolenko bioRxiv2020.04.16.045807;doi:https://doi.org/10.1101/2020.04.16.045807 ShareThisArticle: Copy CitationTools ExtendingthespacingbetweentheShine-DalgarnosequenceandP-sitecodonreducestherateofmRNAtranslocation HironaoWakabayashi,ChandaniWarnasooriya,DmitriN.Ermolenko bioRxiv2020.04.16.045807;doi:https://doi.org/10.1101/2020.04.16.045807 CitationManagerFormats BibTeXBookendsEasyBibEndNote(tagged)EndNote8(xml)MedlarsMendeleyPapersRefWorksTaggedRefManagerRISZotero TweetWidgetFacebookLikeGooglePlusOne SubjectArea Biochemistry SubjectAreas AllArticles AnimalBehaviorandCognition(3592) Biochemistry(7562) Bioengineering(5508) Bioinformatics(20762) Biophysics(10309) CancerBiology(7967) CellBiology(11627) ClinicalTrials(138) DevelopmentalBiology(6602) Ecology(10190) Epidemiology(2065) EvolutionaryBiology(13594) Genetics(9532) Genomics(12834) Immunology(7917) Microbiology(19525) MolecularBiology(7651) Neuroscience(42027) Paleontology(307) Pathology(1254) PharmacologyandToxicology(2196) Physiology(3263) PlantBiology(7029) ScientificCommunicationandEducation(1294) SyntheticBiology(1949) SystemsBiology(5422) Zoology(1114)