生物化学与分子生物学：Lec23-Translation1

(Lostin)TranslationBrieflyreviewprokaryoticmachineryInitiationinEukaryotesWhereintheworldisPeptidyl

Transferase?tRNAcharging:Thesecondcode1968NobelPrizeinPhysiology&Medicine(fordecipheringthegeneticcode)“TriumphoftheChemists”

H.G.KhoranaR.HolleyM.NirenbergUsedacell-freeproteinsynthesissystemfromE.coli,programmeditwithnaturalandsyntheticRNAsofdefinedsequence,anddeterminedthesequenceofthepeptidesproduced.Thecell-freesystemS-30fractionribosomes,tRNAs,tRNAsynthetases,othersolubleproteinfactors20aminoacidsGTP&ATPEnergygeneratingsystemtokeepproducingATPandlimit[ADP]PEP+pyruvatekinasePEP+ADP+Pipyruvate+ATPMg2+andK+(NH4+)TranslationMachineryinProkaryotes(forcomparingwithEukaryotes)

Ribosomes: -70S(composedofL(50S)andS(30S) subunits) -contain23S(L),16S(S),and5S(L)rRNAs -eachsubunit(LandS)contains~30proteinsInitiationfactors:if1,if2,if3Elongationfactors:ef-Tu,ef-Ts,andGTermination(release)factor(s):Rf1andRf2Translationisinitiatedwithfmet(N-formylated methionine).HowisrightAUGselectedfortranslationinProkaryotes?ManymRNAscontainasequenceprecedingthestartcodonthatbase-pairswiththe3'-endof16SrRNA(Shine-Dalgarnosequence) start 5'----GGAGG-------AUG-----3’ mRNA3'----CCUCC--------5' 16SrRNA

Function:helpspositionmRNAinribosome.2. TheAUGitselfisalsoveryimportant3. ThereisaS-Dindependentmodeoftranslation initiationinE.coli4. TranslateinternalORFsofpolycistronicmRNAsS-DTranslationalInitiationinEukaryotesBeginswithmethioninethatisnotformylatedtRNA(tRNAiMet)differentfromtheonethatis usedforinternalmethioninecodonsTranslationstartdeterminedbytheAUGand surroundingsequenceTranslationstartsitealsoaffectedbyRNA structureatthe5’endofthemRNAScanningModelofInitiationProposedbyM.KozakSmallsubunitofribosome(+initiation factors,GTPandtRNAiMet)bindstothe5’Cap,andscansalongthemRNAuntilthefirstAUGTranslationstartsatthefirstAUGModelseemstoworkformostmRNAsScanning(orKozak)ModelforTranslationInitiationinEukaryotesFig.17.16ATPApparentExceptionstotheScanningModel?TranslationofsomemRNAs(5-10%)doesn’t startatfirstAUG(ribosomeskipsoneor moreAUGs)Comparativesequenceanalysisofthese mRNAsrevealedthefollowingconsensus sequenceattheAUGthatisused: -5-4-3-2-1+1+2+3+4

CCRCCAUGG

R=purinePositions-3and+4areparticularlyimportant, basedonmutagenesis

studiesConclusion:WhentheupstreamAUGwasinaweakcontext(likeF9),thenthedownstreamoneisused.Or,putanotherway,thefirstAUGintherightcontextisused.Fig.17.18Effectofthecontextofanupstream“barrier”ATGoninitiationofpreproinsulinmRNA.

proinsulinFig.17.232nded.UpstreamATGisanineffectivebarrieriffollowedbyaStopcodon.InsomemRNAs,thefirstATGisinafavorablecontext,butisstillnotused.KozaknotedthattherewasusuallyaStopcodoninbetweenthestartcodonsinthesemRNAs.SosheengineeredsuchasituationinthepreproinsulinmRNAandtesteditsaffectontranslation.Result:TranslationwasgoodatthedownstreamATGaslongasitwasinagoodcontext.StopcodonConclusionsAnupstreamAUGdoesnotinterfereifit’s context(-3,+4)ispoor,orifitisfollowed quicklybyanin-frameStopcodon.Inthelattercase,itmaybethatthe ribosomesdon’tfalloffthemRNAafter translatingsuchashortORF.InnaturalmRNAs,upstreamORFsare veryshort,unlesstheyhavearegulatory role.Isthefirst“good”AUGreallyfavored?EffectofRepeatedInitiationSequences(replicas)AUGAUGAUGTranslationstartedmainlyatthefirstAUG.Fig.17.19EffectofRNASecondaryStructureinthe5’UTR(Leader)PoorlytranslatedTranslatedwellTrans.wellNottranslatedAdaptedfromFig.17.20ConclusionsSecondarystructure(hairpin)atvery5’endofRNAcanprevent40SsubunitfrombindingScanningribosomescanmeltoutsomehairpins(ΔG=-30kcal/mole),butnothighlystableones(ΔG=-62kcal/mole)InitiatortRNA(tRNAiMet)alsoimportantinrecognizingAUG(yeast)AnticodonoftRNAiMetchangedtoUCC,translationstartedatfirst“good”AGGinhis4mRNA(Fig.17.21).Fig.17.22SummaryoftranslationinitiationinEukaryotes.Resistsbindingto60SsubunitInitiationFactors

(excepteIF-4)eIF-1(and1A):promotesscanning*eIF-2:bindstRNAiMetto40Ssubunit,requires GTP(whichgetshydrolyzedtoGDP)eIF-2B:catalyzesexchangeofGTPforGDPon eIF-2*eIF-3:bindsto40Ssubunit,prevents60Ssubunit frombindingtoiteIF-5:stimulates60Ssubunitbindingtothe48S pre-initiationcomplex*eIF-6:bindsto60Ssubunit,helpsprevent40S subunitfrombindingtoit*

prokaryoticcounterparteIF4(eIF4F)eIF4F

OriginallyisolatedbasedonitsabilitytobindtheCap-nucleotide7MeGTP.Composedof3subunits,a24-kDaproteinthatbindstheCap,and2othersthatstabilizethecomplexandhaveotherroles:eIF4G-versatileadaptoreIF4A-RNAhelicaseeIF4E-bindstheCapFig.17.25eIF4AandeIF4BeIF4AalsoexistsoutsideoftheeIF4FcomplexcontainsaDEADmotif(aspartate-glutamate-alanine-aspartate)characteristicofRNAhelicasesRNAhelicaseactivitywasdemonstrated(rightpanel)andfoundtorequireATPandtobestimulatedbyanotherprotein,eIF4BeIF4B

bindsRNA,stimulateseIF-4ARoleintranslation:Unwindhairpinsinthe5’UTRs17.26eIF4G–helpsrecruit40SsubunittomRNA;caninteractwitheIF4E,eIF4A,eIF3,andpoly-Abindingprotein(Pab1);mayberesponsibleforthesynergisticeffectofCapandpolyA-tailontranslation.WhyinteractwithbothCapandpolyA-tail?Similarto17.27cObservation:SomeviralmRNAs(suchasPoliovirus)arenotcapped,yetarepreferentiallytranslated.Somearealsotranslatedviainternalribosomeentrysites(IRES)(apparentlywithoutscanningtothem).Mechanism:ViralproteaseclipsoffN-terminusofeIF4G,soitcan’tbindeIF4E.eIF4Gbindsaviralprotein(X),thatbindstotheIRES