Chapter 18 elongation and termination

Chapter18TheMechanismofTranslationII:ElongationandTermination18.1TheDirectionofmRNATranslationProteinchainsgrowintheamino-to-carboxyldirection.proteinchainsareinallstagesofcompletionwhenthelabeledaminoacidisadded.IfithadstartedattheN-terminus,thentheC-terminalpeptideswouldbethemosthighlylabeledN-terminalpeptideC-terminalpeptideN-terminalpeptideC-terminalpeptide18.2TheGeneticCode1.NonoverlappingCodons2.NoGaps(commas)intheCode3.TheCodeistripletInmostcases,morethanonetripletcodesforagivenaminoacid.4.TheCodeisdegenerate5.UnusualBasePairsbetweenCodonandAnticodonwobblehypothesis：Thefirsttwobasesofacodon←→theanticodon.ThewobblephenomenonreducesthenumberoftRNAsrequiredtotranslatethegeneticcode:Thelastbaseofthecodon“wobble”theanticodon.Baseatfirstposition(5’end)oftRNABaseatthirdposition(3’end)ofmRNAAUCGGCorUUAorGI(Inosine)A,UorCinosine(I)isananalogofG5.TheCodeis(Almost)UniversalDeviationsfromthe“Universal”GeneticCodeTripletNormalcodeAlteredcodeSourceUGATerminationTryptophanHumanandyeastmitochondria;mycoplasmaCUALeucineThreonineYeastmitochondriaAUAIsoleucineMethionineHumanmitochondriaAGAAGGArginineTerminationHumanmitochondriaUAATerminationGlutamineParameciumTetrahymena;StylonychiaUAGTerminationGlutamineParamecium18.3TheElongationMechanism1.AThree-SiteModeloftheRibosomeEPAsmallsubunitlargesubunit2.ElongationStep1BindinganAminoacyl-tRNAtotheASiteoftheRibosomeThreeelongaionfactors(EF):EF-TuEF-TsEF-GTransferaminoacyl-tRNAstotheribosomeGTPaseactivity（translocation)AllneedGTPGTPwasneededGTPwasnecessaryforEF-T-dependentbindingofPhe-tRNAPhetotheribosomes.PolymerizationrequiredbothEF-TandEF-GandahighconcentrationofGTPRequirementforBothEF-TsandEF-TutoBind[3H]Phe-tRNAtoRibosomesCarryingPreboundN-acetyl-[14C]Phe-tRNAAdditions[3H]Phe-tRNAbound(pmol)None2.8EF-Ts+GTP2.8EF-Tu+GTP5.2EF-Ts+EF-Tu+GTP11.6EF-TuandEF-Tscooperatetocausetransferofaminoacyl-tRNAstotheribosome.PAPAPATheribosomehasfMet-tRNAfMetinthePsiteandanaminoacyl-tRNAintheAsite.3.ElongationStep2PeptideBondFormationThepuromycinreactionasanassayforpeptidyltransferase50Sribosomalsubunit,withoutanyhelpfromthe30Ssubunitorsolublefactors,couldcarryoutthepeptidyltransferasereactionThe23SrRNAcatalyzethepeptidyltransferasereactionSDSandproteinaseKdidnotdestroy50Speptidyltransferase.PhenolcoulddestroyE.coli,butnotT.aquaticusPhenoldisruptingsomehigher-orderRNAstructure.RNasedestroypeptidyltransferaseofT.aquaticusPositivecontrol4.ElongationStep3TranslocationTomovethemRNAandpeptidyl-tRNAonecodon’slengththroughtheribosome.18.4Termination2.ReleaseFactors(RFs)1.StopCodonsUAA,UAG,UGAtRNAsdonotordinarilyrecognizestopcodons.RF-1:UAGRF-2:UGARF-1orRF-2：UAARF-3:resemblestructureoftRNAGTP-bindingproteinthatfacilitatesbindingofRF-1and-2totheribosomeThreeaminoacidsinRFbindtostopcodonorAorAUAGorUAAUGAorUAAUUEukaryotesRFsEukaryoteshavetworeleasefactors:eRF1:recognizesallthreeterminationcodons.eRF3:aribosome-dependentGTPasethathelpeRF1releasethefinishedpolypeptide.