ProteinSynthesisTranslation教学讲解教学课件

ConceptoftranslationProteinsynthesisrequires:

•mRNA、rRNA、tRNA•Substrates:20aminoacids•Enzymesandproteinfactors:initiationfactor,elongationfactor,releasingfactor•ATP、GTP、Mg2+ProkaryoticmRNAispolycistron.EukaryoticmRNAismonocistron.TheGeneticCode

TheGeneticCodeIsaTripletCode

64,morethanrequired,butnotoverlyexcessive

Threecodonsforstopsignal:UAA,UAG,UGA.61codonsfor20aminoacids.Onecodonforstartsignal:AUG.Italsocodesformethionine.NonoverlappingandwithoutPunctuation

OpenreadingframeFrameshiftDegenerate,butUnambiguous

Synonymouscodons?

Morefault-tolerantforpointmutations.

WobbleBasepairsofinosineFewerthan61tRNAsarerequiredEnhancestheefficiencyofproteinsynthesis

TheCodeIsAlmostUniversalAllorganismsusethesamegeneticcode.CytoplasmAUA:IleAUG:Met,initiationUAA,UAG,UGA:terminationMitochondriaAUA:Met,initiationUGA:TrpAGA,AGG:terminationHowever,thereisintriguing

exceptionofafewminorvariations.tRNAandAAActivationtRNAActivationofaminoacidActivatedaminoacidAla-tRNAAlaSer-tRNASerMet-tRNAMetSummaryofAAactivationActiveformaminoacyl－tRNAActivationsite

α-carboxylgroupLinkageesterbondActivationenergy2high-energybondsProteinsynthesisfidelityAminoacyl-tRNAsynthetasehastheproofreadingability.TheoverallerrorratefortranslationinE.coliisonly1per50,000codons.ProkaryoticMet-tRNAmetProkaryoticMet-tRNAmetcanbeformylatedtofMet-tRNAimet.Met-tRNAmet+N10-formyltetrahydrofolate

FormyltransferasefMet-tRNAimet+tetrahydrofolate

Initiationcodons

Forprokaryotes:fMet-tRNAimetinitiationMet-tRNAemetelongation.

Foreukaryotes:Met-tRNAimetinitiation.Met-tRNAemetelongation.Recognizedbyenzymesorfactorsindifferentphases.RibosomeofprokaryotesThreesitesonribosomesLocationFunctionAminoacylsite(Asite)ComposedbylargeandsmallsubunitAcceptinganaminoacyl-tRNAPeptidylsite(Psite)ComposedbylargeandsmallsubunitFormingthepeptidylbondsExitsite(Esite)OnlyonlargesubunitReleasingthedeacylatedtRNAAsite,PsiteandEsite

Theaminoacyl(A)site,thepeptidyl(P)site,andtheexit(E)site

TranslationinProkaryotes

InitiationThreestepsinprokaryoticsystems–PositioningmRNAonthe30Ssubunit–RegisteringfMet-tRNAimetonthePsite–Associatingthe50SsubunitThreeinitiationfactors:IF-1,IF-2andIF-3.Inprokaryotes,thetwotypesoftRNAspecificformethioninearedesignatedtRNAfMetandtRNAMet.ThetRNAfMetisusedfortheAUGinitiationcodonandiscalledtheinitiatortRNA.Shine-Dalgarnosequenceaninitiationsignalofpurine-rich

The3′endof16srRNAhasconsensussequence

UCCU

whichiscomplementarytoAGGAinS-Dsequence(alsocalledribosomalbindingsite).Initiation1TheIF-1andIF-3bindtothe30Ssubunit.ThemRNAthenbindstothissubunit.The16SrRNAisalignedwithmRNA,allowingAUGonthePsite.Initiation2ThecomplexoftheGTP-boundIF-2andthefMet-tRNAentersthePsite.Initiation3The50Ssubunitcombineswiththiscomplex.GTPishydrolyzedtoGDPandPi.AllthreeIFsdepartfromthiscomplex.Notethat:unlikeallotheraminoacyl-tRNAmoleculeswhichbindtheAsite,thebindingoffMet-tRNAfMetoccursdirectlyintothePsite.

ElongationThreestepsineachcycle:–Positioninganaminoacyl-tRNAintheAsite–Formingapeptidebond–TranslocatingtheribosometothenextcodonElongationfactors(EF)arerequired.Ribosomalcycle:ThepeptidechainisextendedbyoneaminoacidastheribosomemovesonecodondownstreamonmRNA.TheelongationcyclescontinuesuntilthestopcodonentersintotheAsite.Step1:EntranceAnAA-tRNAoccupiestheemptyAsite.RegistrationoftheAA-tRNAisanenergyconsumingprocess.TheentranceofAA-tRNAneedstoactivateEF-T.TheenergyisprovidedthroughahydrolysisofGTP.EFactivationcycleEF-TiscomposedofTuandTssubunits.Tuhastwoactivesites,onefor

GTP

andanotherfor

AA-tRNA.Tuis

GTPase.TucannotbindtotheinitiatingAA-tRNA.Peptidebondformation1Peptidebondformation2PeptidyltransferaseisrequiredTranslocation1EF-Gisa

translocase.GTPboundEF-GprovidestheenergyTranslocation2Thedipeptidyl-tRNAisnowentirelyinthePsite,andtheAsiteisopenforthenextincomingAA-tRNA.TerminationProkaryoteshave3releasefactors:RF-1,RF-2andRF-3.–RF-1andRF-2:Recognizingtheterminationcodons–RF-3:GTPhydrolysisandcoordinatingRF-1/RF-2andrpsTermination1UAAandUAG:RF-1;UAAandUGA:RF-2.Termination2The

peptidyl

transferaseisconvertedtoan

esterase.Theesterbondofpolypeptidyl-tRNAatthePsiteishydrolyzed,andthenascentpeptideisreleased.Termination3TheunchargedtRNA,mRNA,andRFsdissociatefromtheribosome.Thewholeprocessofproteinsynthesisfinishes.EnergyconsumptionInitiation：oneGTP(IF-2-GTP)AAactivation：two~Pbondselongation：twoGTP(Tu-GTP,EF-G-GTP)Termination：oneGTP(RF-3)PolysomeProteinsaresynthesizedonasinglestrandmRNAsimultaneously,allowinghighlyefficientuseofmRNA.AnimationInterferenceofTranslationTheproteinsynthesisishighlyregulated.Thisprocesscanalsobetheprimarytargetformanytoxins,antibioticsandinterferons.TheseinterferantsinteractspecificallywithproteinsandRNAstointerrupttheproteinsynthesis.Theinterferenceleadstothedeathofbacteriaforclinicaltreatment.AntibioticsNameTargetFunctionTetracycline30SblocktheAsitetopreventbindingofAA-tRNAwith30SStreptomycin30SrepressthetranslocaseChloromycetin50Sblockthepeptidyltransferase,andinhibittheinitiationErythromycin50SInhibitthetranslocaseCycloheximide60Srepressthetranslocase,inhibittheelongationPuromycinreleasetheprematuredpeptideChemicalstructuresPuromycinAminoacyl-tRNAanalog,bindsatAsiteandactsaspeptidylacceptor,abortingpeptideelongation.Itworksforbothprokaryotesandeukaryotes.ToxinsOthertoxins,suchasplantproteinRicin,isamongthemosttoxicsubstanceknown,whichactson60ssubunits.Theyapparentlycatalyzesomemodificationofaribosomalproteinbutthechemicalnatureofthealterationisunknown.DiphtheriatoxinInterferonTheinitiationisarate-limitingstep.Biologicallyactivebiomoleculesaffecttheproteinsynthesis.Interferonsarecytokinesproducedduringimmuneresponsetoantigens.InterferonProteinModificationand

ProteinTargeting

ProteinFoldingModificationofprimarystructureRemovalofthethefirstN-terminalmethionineresidueCovalentmodification

ofsomeaminoacids(phosphorylation,methylation,acetylation,…)Activationofpeptidesthrough

hydrolysisModificationofspatialstructureAssembleofsu