生理化学教学课件：Protein Synthesis

ProteinSynthesis

(Translation)ProteinSynthesisSystem

(Componentsrequiredfortranslation)SectionOnetRNAistheadaptorintranslationanticodonloopaminoacidarmcloverleafAminoacid+tRNAaminoacyl-tRNAATPAMP＋PPi(Pyrophosphate)aminoacyl-tRNAsynthetaseAminoacidisactivatedbyanesterbondwiththe3’terminusoftRNA1ststep:Aminoacid＋ATP＋E

aminoacyl-AMP-E＋PPi目录AminoacidAminoacyl-AMPAACarboxlyPhosphate2ndstep:aminoacyl-AMP-E＋

tRNA↓

aminoacyl-tRNA＋AMP

＋

E目录AAAAminoacyl-AMPaminoacyl-tRNAsynthetaseAminoacyl-tRNA（AA-tRNA）Aminoacyl-tRNAsynthetasescatlyzespecificaminoacidsandtRNAs(20types).aminoacyl-tRNAsynthetasehasproof-readingactivity(editingactivity);errorrate1/40,000aminoacyl-tRNA(AA-tRNA)：Lys-tRNALysMet-tRNAMet

Activeform

aminoacyl－tRNAActivationsite

a-carboxylgroupLinkage

esterbondActivationenergy

2high-energybondsSummaryofAAactivation

RibosomesRibosomeistheworkbenchwhereproteinsynthesistakesplace.Aribosomeiscomposedofalargesubunitandasmallsubunit,eachofwhichismadeofribosomalRNAsandribosomalproteins.MolecularcomponentsofribosomeofprokaryotesS=sedimentationcoefficient目录Prokaryoteribosomeeukaryoteribosome70S80S（120nucleotide）（3200nucleotide）>30proteins5SrRNA（120nucleotide）28SrRNA（4700nucleotide）5.8SrRNA（160nucleotide）~49proteins>20proteins~33proteins（1540nucleotide）18SrRNA（1900nucleotide）

Aminoacylsite(Asite)LargeandsmallsubunitsAcceptinganaminoacyl-tRNAPeptidylsite(Psite)LargeandsmallsubunitsFormingthepeptidylbondsExitsite(Esite)LargesubunitReleasingthedeacylatedtRNAlocationfunctionThreeimportantsitesonribosomes

Asite,PsiteandEsiteThefunctionofribosomeinproteinsynthesisToholdmRNA,aminoacyl-tRNAandtranslationfactorsinrightplaceforproteinsynthesis.TocatalyzecertainchemicalreactionsintheprocessofproteinsynthesisProteinSynthesisProcessSectionTwo2.1FormationofInitiationComplexIntheinitiationstage,ribosome,initiationaminoacyl-tRNAandmRNAmoleculesareassembledtothetranslationalinitiationcomplex

Forprokaryotes:fMet-tRNAimetcanonlyberecognizedbyinitiationcodon.（甲酰甲硫氨酸）Met-tRNAemetisusedforelongation.Foreukaryotes:Met-tRNAmetisusedforbothinitiationandelongation.（甲硫氨酸）InitiationtRNAProkaryotic

Met-tRNAmetcanbeformylatedtofMet-tRNAimet.Prokaryotic

Met-tRNAmetMet-tRNAmet+N10-formyltetrahydrofolate(四氢叶酸)fMet-tRNAimet+tetrahydrofolateformylTransferase甲酰转移酶甲酰化TranslationalinitiationfactorsinprokaryoteIF-3

isneededfor30SsubunitstobindspecificallytoinitiationsitesinmRNA.

IF-2

bindsaspecialinitiatortRNAandcontrolsitsentryintotheribosome.

IF-1

bindsto30Ssubunitsonlyasapartofthecompleteinitiationcomplex,andcouldbeinvolvedinstabilizingit,ratherthaninrecognizinganyspecificcomponent.具体功能见书P374表格theformingoftranslationalinitiationcomplexinprokaryoteInitiationfactorsIF-1

andIF-3bindtothefree30ssubunit；mRNAbindtoribosome30ssubunit；Withthehelpof

IF-2

andGTP,theinitiatorfMet-tRNAcanbindtotheinitiationcodon(AUG)onthemRNA；The50Ssubunitbinds,displacingIF1,IF2andIF3.The70Sinitiationcomplexisformed.配合书上P375的图看S-Dsequence（Shine-Dalgarnosequence）,alsocalled（ribosomalbindingsite,RBS）

8-13ntupstreamoftheinitiationcodoninprokaryoticmRNAwhichbase-pairswithacomplementarysequencenearthe3’endof16SrRNA.ThatresultsintheinitiationcodoninthePsite.S-Dsequence

rpS-1recognizingsequence16S-rRNAinribosomesmallunitDifferencesofinitiationbetweeneukaryoteandprokaryote3stepsofeukaryoticinitiationcomplexformation：43Spre-initiationcomplexformation48Spre-initiationcomplexformation80SinitiationcomplexformationEukaryoticinitiationfactorsFactorFunctioneIF2B,eIF3Firstfactorstobind40Ssubunit;facilitatesubsequentstepseIF2FacilitatesbindingofinitiatingMet-tRNAMetto40SribosomalsubuniteIF4ARNAhelicaseactivityremovessecondarystructureinthemRNAtopermitbindingto40Ssubunit;partoftheeIF4FcomplexeIF4BBindstomRNA;facilitatesscanningofmRNAtolocatethefirstAUGeIF4EBindstothe5’capofmRNA;partoftheeIF4FcomplexeIF4GBindstoeIF4Eandtopoly(A)bindingprotein(PAB);partoftheeIF4FcomplexeIF5PromotesdissociationofseveralotherIFsfrom40Ssubunitasapreludetoassociationof60Ssubunittoform80SinitiationcomplexeIF6Facilitatesdissociationofinactive80Sribosomeinto40Sand60SsubunitsMet40SMetMet40S60SmRNAeIF-3①elF-2-GTP②GDP+PiReleaseeIFselF-5④ATPADP+PielF4E,elF4G,elF4A,elF4B,PAB③MetMet-tRNAiMet43Spre-initiationcomplex48Spre-initiationcomplex80StranslationalinitiationcomplexEukaryotictranslationalinitiationcomplexformation

2.3ElongationThreestepsineachcycle:Positioninganaminoacyl-tRNA(AA-tRNA)intheAsite---

EntranceFormingapeptidebond---Peptidebondformation

Translocatingtheribosometothenextcodon---TranslocationElongationisaprocessofrepeatedribosomalcyclesofaminoacidaddition.Polypeptidesgrowstepwisefromtheaminoterminustothecarboxylterminus.Elongationfactors(EFs)arerequiredProkaryote：EF-Tu,EF-TsandEF-GEukaryote：eEF-1α,eEF-1βandEF-2Step1:EntranceAnAA-tRNAoccupiestheemptyAsite.RegistrationoftheAA-tRNAconsumeoneGTP.TheentranceofAA-tRNAneedstoactivateEF-T.Step2:PeptidebondformationThepeptidebondformationoccursattheAsite.Theformylmethionylgroupistransferredtoα–NH2oftheAA-tRNAattheAsitebyapeptidyltransferaseThe23SrRNA(ineukaryoticribosomethe28SrRNA)catalyzesthepeptidebondformation:ribozymeStep3:TranslocationEF-Gisatranslocase.GTPboundEF-Gprovidestheenergytomovetheribosomeonecodontowardthe3’endonmRNA.Afterthetranslocation,theunchargedtRNAisreleasedfromtheEsite.

2.4TerminationReleasefactorsProkaryotesRF-1recognizestheterminationcodonsUAAandUAGRF-2recognizesUAAandUGARF-3isaGTP-bindingproteinthatleadstothedissociationofRF1/RF2afterpeptidereleaseEukaryoteseRF1recognizesallthreeterminationcodonseRF3isaribosome-dependentGTPasethathelpseRF1releasethecompletedpolypeptideTermination1TheunchargedtRNA,mRNA,and