生化2014c17proteintransla

Biochemistry,Dept.ofBiochemistryandMolecularBiology,ProfessorWuYaosheng2014-06,Chapter17,ProteinBiosynthesis,ProteinBiosynthesis,Genes,Geneexpression,?,AGCT,AGCU,20种AA,4,Questions,1.Whatarerequirementsforoneproteinbiosythesisineukaryotecells?,4.Studieshaveshownthattheoccurrenceofadiseaseassociatedwithabnormalchangesofthestructureofaproteininhepatocytes.Iask,howtoidentifythisproteinstructurehaschanged?Whethertoputforwardyourideas?,3.Howtosynthesizeamoleculeofhemoglobinwithbiologicalfunctionsbygeneexpressionprocess?,2.HowmanysequencesofDNAcouldcodeforthefragmentofonepeptideGlu-Glu-Met-Trp-Asp？,5,Section1ProteinBiosynthesisSystem,MajorTopics,Section2LinkageofAminoAcidswithtRNA,Section4ModificationandTargetTransportationofPost-translation,Section5InterferenceandInhibitionofProteinBiosynthesis,Section3BiosynthesisProcessofPolypeptidechain,6,KeyPoints,FunctionsofmRNA,tRNA,rRNAduringPrsynthesis,geneticcodoncharactersActioncharactersofaminoacyl-tRNAsynthetaseComparisonofthetranslationprocessinprokaryoteandineukaryoteMolecularchaperoneandmodificationtypespost-translationSignalpeptidesandtheiractions,directionaltransportationcharacteristicsMechanismoftranslationinhibitionbyantiboitics,toxins,andinterferons,7,BiologicalSynthesisProcessofProtein,Theactivationandtransferofaminoacids,Thesynthesisofpolypeptide,Themodificationofpost-translation,Initiation,Elongation,Termination,Entryofaminoacyl-tRNA,Formationofpeptidebond,Translocationofribosome,Ribosomalcycle,8,SectionOne,ProteinBiosynthesisSystem,Distinguishmonocistronandpolycistron;Wobble,Degeneracyofcodons,Keypoints,FunctionsofmRNA,tRNA,ribosome;ORF,codonsandtheircharacters,Difficulties,10,RequirementsofProteinBiosynthesis,VariousRNAs:mRNA、rRNA、tRNA,Substrate：aminoacids,Proteinfactors:Initiationfactors,Elongationfactors,Terminationfactors,Enzyme:aminoacyl-tRNAsynthetasepeptidyltransferase,Energysupply:ATP,GTP,Inorganicion:Mg2+,11,1.MessengerRNA(mRNA)andItsCodes,MessengerRNAisapolycistroninprokaryotes,butamonocistronineukaryotes,Proteins,Protein,Function:Asatemplateofproteinsynthesis,Cistron:encodeonepolypeptide,12,Conceptofcodons,ThegeneticcodeisthesetofrulesthatspecifyhowthenucleotidesequenceofanmRNAistranslatedintotheaminoacidsequenceofapolypeptide.,EachaminoacidinaproteinisrepresentedbythreeconsecutivebasesinmRNA,andthesethree-basecombination,ortriplets,arecalledcodons.,13,Thegeneticcodeconsistsof64tripletcodons(A,G,C,U)，43=64,Termination(stop)codons:,Startcodon：,UAA,UAG,UGA,AUG(methionine),14,TheGeneticCode,UUUUUCUUAUUGCUUCUCCUACUGAUUAUCAUAAUGGUUGUCGUAGUG,UCUUCCUCAUCGCCUCCCCCACCGACUACCACAACGGCUGCCGCAGCG,UAUUACUAAUAGCAUCACCAACAGAAUAACAAAAAGGAUGACGAAGAG,UGUUGCUGAUGGCGUCGCCGACGGAGUAGCAGAAGGGGUGGCGGAGGG,PhenylalanineLeucineLeucineValine,IsoleucineMethionine,SerineProlineThreonineAlanine,TyrosineStopHistidineGlutamineAsparagineLysineAsparticacidGlutamicacid,CysteineArginineSerineArginineGlycine,StopTryptophan,15,CharacteristicsofcodonsonmRNA,(1)Direction（方向性）,(2)Commaless(连续性）,(3)Degeneracy(简并性）,(5)Wobble（摆动性）,(4)Universality（通用性）,16,ReadingframeReadingframeisdeterminedbytheAUGinitiationcodon.Everysubsequenttripletisreadasacodonuntilreachingastopcodon.,MetAlaGluTrpLeuSerMetSerstop,17,18,HemoglobinWayne(3terminalframeshiftmutation),Normal-globinACGUCUAAAUACCGUUAAGCUGGAGCCUCGGUA.ThrSerLysTyrArgstop,mutatedregion,Wayne-globin.ACGUCAAAUACCGUUAAGCUGGAGCCUCGGUAGThrSerAsnThrValLysLeuGluProArgstop,19,Degeneracy,Mostaminoacidshavemorethanonecodon,meansdegenerate.Codonsthatspecifythesameaminoacidoftendifferonlyinthethirdbase.,AmutationonlywithabasechangeinDNAmaynotresultinaminoacidchangeintheencodedprotein.,CUACUG(LeuLeu)withoutaffectingtranslation,20,Degeneracy(简并性),AminoacidNo.ofcodonAminoacidNo.ofcodon,21,Universality,Thegeneticcodeisthesameinmostorganisms.,Exceptionsarefoundinmitochondrialgenome,wheresomecodonsspecifydifferentaminoacidstothosenormallyencodedbynucleargenes.,Insomemitochondria,theUGAcodonnolongerspecifiesterminationoftranslationbutinsteadencodestryptophan.,22,23,Preference,Leucinecodons(89)ofribosomeproteininE.coliTotal1209codonsUUAUUGCUUCUCCUACUG1243079,23,Wobble,Thefirstbaseontheanticodonalwayspairswiththethirdbaseonthecodonandnotstrictlyobeystherulesofbase-pairing.,Codon-anticodonbase-pairingisantiparallel.,Codon-anticodoninteractions,OnetRNAcaninteractwithmorethanonecodon(therefore50tRNAs).,Thethirdpositioninthecodonisfrequentlydegenerate.,24,25,U,摆动配对,Wobblepairingofanticodonwithcodon,26,onetRNAleucanreadtwooftheleucinecodons,27,tRNAisthe“adaptor”moleculeinproteinsynthesis.,2.TransferRNA(tRNA),Twofunctionalsites:(1)acceptorstemCCA-OH3terminuscancarryaminoacidonterminaladenosine.(2)anticodonstemandanticodonloopTorecognizethecodononmRNA,28,(1)acceptorstem,(2)anticodonstemandanticodonloop,ThesecondarystructureoftRNA,29,DHUarm,Anticodon,Acceptorarm,arm,30,tRNA,31,prokaryoticribosome,eukaryoticribosome,3.rRNAsandRibosomes,32,Ribosomeswasorganizedinthreeways,PolysomesseveralribosomesbindtoandtranslateasinglemRNAsimultaneouslyFreeribosomesSynthesizeproteinsofcytosol,nucleus,mitochondriaMembraneboundribosomesSynthesizesecretedproteinsormembraneprotein,33,polypeptidechain,aminoacids,Largesubunit,Thefunctionsofribosome:Fortheassemblyofaminoacidstobeapolypeptidechain,34,Questions,1.Degeneratecodonsdiffermostlyin,A.theidentitiesoftheirfirstbase.B.theidentitiesoftheirsecondbase.C.theidentitiesoftheirthirdbase.D.thewobbleposition.E.Choicesthreeandfourarebothcorrect.,35,35,Questions,2.RibosomesselectthecorrecttRNAs,A.andthenchargethemusingtheappropriatesynthetase.B.thenbindtotheappropriatemRNA.C.solelyonthebasisoftheiranticodons.D.withtheleastabundantanticodons.E.dependingontheirabundanceinthecytosol.,36,Questions,3.Degenerateofgeneticcodonmeansthat,A.1AAhasmorethan2codonsB.2codonscouldbecombinedas1codonC.eachaminoacidalwayshasseveralcodonsD.1codoncanrepresentdifferentaminoacid.E.1aminoacidonlyhas1codon.,37,Questions,4.TheanticodonWhichcanpairtothecodonCGAonmRNAis,A、GCUB、UCGC、CCAD、UCUE、ACU,38,SectionTwo,LinkageofAminoAcidwithtRNA,Theactivationandtransferofaminoacids,Aminoacyl-tRNAsynthetase,Twospecificities,TocatalyzetheformationofesterbondbetweentRNAandaminoacidToproofreadthewrongesterbond,Twoactivities,TorecognizetheaminoacidTorecognizethetRNA,40,Aminoacid+ATP,Aminoacyl-AMP,PPi,Aminoacyl-tRNAsynthetase,41,Aminoacyl-AMP+tRNA,Aminoacyl-tRNA+AMP,42,Ala-tRNAAla,Gly-tRNAGly,fMet-tRNAiMet,Met-tRNAiMet,Formylatedmethionine,Twohighenergyphosphatebondsneeded,43,TheformationoffMet-tRNAimet:,Metineukaryotesisnotformylated,Encodedbyinitiationcodoninprokaryotesisformylated(甲酰化）,44,Questions,1.HydrolyticeditingofincorrectlychargedtRNAs,A.involvesattackofPPiontheaminoacyl-AMPintermediate.B.occursatasecondactivesiteonthesynthetase.C.isawasteofATP,thatcouldbeusedforothercellularfunctions.D.isanecessarystepforalloftheaminoacyl-tRNAsynthetases.E.occurswhentheincorrecttRNAbindstothesynthetase.,45,Questions,2.MostoftheidentityelementsforcorrectaminoacylationoftRNAsarefound,A.intheanticodonstemandthe3CCAendB.intheTLoopandtheDstemandloop.C.intheanticodonandtheacceptorstem.D.solelyintheanticodon.E.distributedthroughoutthestructure.,46,SectionThree,BiosynthesisProcessofPolypeptidechain,BiologicalSynthesisProcessofProtein,Theactivationandtransferofaminoacids,Thesynthesisofpolypeptide,Themodificationofpost-translation,Initiation,Elongation,Termination,Entryofaminoacyl-tRNA,Formationofpeptidebond,Translocationofribosome,Ribosomalcycle,48,1.Theinitiationofpolypeptidesynthesis,Toformaninitiationcomplex,IF1,IF2,IF3inprokaryotesMorethan9eIFineukaryotes,Initiationfactors(IF):,49,ProkaryoticandEukaryoticinitiationfactors,50,TheInitiationofTranslationinProkaryotes,Step1Dissociation(解聚)ofribosomesubunits,Step2mRNAentersthesmallsubunit,Step3BindingoffMet-tRNAifMet,Step4Bindingoflargesubunit,51,IF-3,IF-1,Step1Dissociation(解聚)ofribosomesubunits,52,IF-3,IF-1,Step2mRNAentersthesmallsubunit,53,MoleculeinteractionbetweenmRNAandthesmallsubunitinprokaryotes.,16s-rRNAofsmallsubunit,mRNA,SD：Shine-Dalgarno:ribosomalbindingsite,RBS,54,IF-3,IF-1,Step3BindingoffMet-tRNAifMet,55,IF-3,IF-1,IF-2,GTP,GDP,Pi,Step4Bindingoflargesubunit,56,IF-3,IF-1,IF-2,-GTP,GDP,Pi,57,TheInitiationofTranslationinEukaryotes,Step1Dissociation(解聚)ofribosomesubunits,Step3mRNAentersthesmallsubunit,Step2BindingofMet-tRNAiMet,Step4Bindingoflargesubunit,58,eIF-3,eIF-2B,60S,40S,eIF-6,Step1Dissociation(解聚)ofribosomesubunits,59,eIF-1,eIF-6,40S,eIF-3,Step2BindingofMet-tRNAiMet,60,eIF-6,40S,eIF-3,Step3mRNAentersthesmallsubunit,61,40S,Met,5cap,3PolyA,Stopcodon,AUG,60S,Step4Bindingoflargesubunit,62,2.Theelongationoftranslation,Toelongatethepolypeptidechain,aserialsofcyclescalledribosomalcyclesareneeded,Theentryofaminoacyl-tRNA:oneGTPneeded,Theformationofpeptidebond:peptidyl-transferaseneeded,Thetranslocation:oneGTPneeded,Including:,63,Theelongationfactorsoftranslation,ProkaryoteFunctionsEukaryote,EF-Tu,tohelpaminoacyl-tRNAentryAsite,EF-Ts,tobindwithGTP,EF-G,EF2,Translocase,helpthemovementofpeptidyl-tRNAfromAsitetoPsite,regulatorysubunits,EF-1-,EF-1-,64,ElongationProcessinProkaryotes,Toproteinelongationcartoonmovie,65,Elongationprocessineukaryotes,ItisidenticaltothatinprokaryotesdespiteofthedifferentelongationfactorThepolypeptidechainiselongatedbyadditionofaminoacidresiduesonebyoneonitscarboxylendwiththerelativemovementoftheribosomeonmRNAcodonbycodon,in53direction.,66,3.Theterminationoftranslation,Toreleasethepolypeptidechainfromtheribosome.,TountietheribosomefrommRNA,tRNA,andtoseparatethelargesubunitfromsmallsubunit.,Theribosomecanbereusedforanotherbiosynthesisprocessofpolypeptide.,67,Termination,RF-3：PromotethebindingofRF-1orRF-2toribosome;GTPaseactivity,RF-1：UAA，UAG,RF-2：UAA，UGA,WhenterminationcodonisontheAsite,RFrecognizesitandstarttheterminationprocess.,Releasefactorineukaryote:eRF,eRFrecognizesallofthe3termination,68,TerminationofProteinBiosynthesisinProkaryotes,69,RF,Releasefactors,Hydrolaseactivity,70,Terminationoftranslation,Eukaryote,Terminationineukaryotesiscatalyzedbyasingleeukaryoticreleasefactor.,Allprocessoftranslationterminationineukaryoteissimilartothatinprokaryoteinmanyways.,71,Terminationoftranslation,DirectionofproteinsynthesisisNterminustoCterminus.,Directionoftranslationis5to3alongthemRNA.,72,Energyconsumptioninproteinsynthesis,1.Activationofaminoacid：2ATP,2.elongation：2GTPperpeptidebond,A.Entrance：1GTP,Foreachpeptidebondformation,fourhigh-energybondsarehydrolyzed.,B.Translocation：1GTP,73,Thesignificanceofpolysomes,Toenhancethevelocityofsynthesisofpolypeptidechain.,Polysomes,Duringproteinsynthesis,asinglemRNAmoleculeisboundbymultipleribosomesinintervalofabout100nucleotides,andpolypeptidesbeingsynthesizedsimultaneously.ThisstructureofmRNAwithmultipleribosomesattachediscalledpolysomes.,Direction,74,Polysomesseenwithelectronmicroscope,75,Questions,1.ThepathwayofatRNAduringpolypeptideelongationontheribosomeis,A.Asite-Psite-Esite.B.Psite-Asite-Esite.C.Asite-Psite-entrysite.D.Psite-entrysite-exitsite.E.Noneofthechoicesarecorrect.,76,Questions,2.Ontheribosome,mRNAbinds_;thepeptidyltransferasereactionoccurs_.,A.betweenthesubunits;onthelargesubunit.B.betweenthesubunits;onthesmallsubunit.C.tothelargesubunit;onthesmallsubunit.D.tothesmallsubunit;onthelargesubunit.E.tothesmallsubunit;betweenthesubunits.,77,Questions,3.Inprokaryotes,proteinsynthesisratesarelimitedbytherateofmRNAsynthesis.RNAsynthesisoccursatabout50nucleotides/sec.Therefore,proteinsynthesisoccursatabout_aminoacids/sec.,A.10B.17C.25D.34E.50,78,SectionFour,Post-translationProcessingandTargeting,Post-translationProcessing,(2)Modificationofproteinprimarystructure,(1)Foldingofnewlysynthesispolypeptide,(3)Thepolymerizationofsubunits,(4)Targettransportationofproteins,80,(1)Foldingofnewlysynthesispolypeptide,A.Molecularchaperones,B.Isomerases,heat-shockprotein(HSP)family,Chaperone,chaperonin,Proteindisulfideisomerase(PDI),Peptidylprolylcis-transisomerase(PPI),81,(2)Modificationofproteinprimarystructure,hydrolyticmodification,Acylation,hydroxylation,methylationorphosphorylationofpolypeptide.,RemovingofMetresiduesorafewofaminoacidresiduesontheN-endterminusofproteins.,Glycosylationofproteins,Modificationofproteinonsidechainstructures,N-endmodification,82,N,C,signalpeptide,KR,KR,POMC(鸦片促黑皮质素原),pro-opiomelanocortin,istheprecursorofseveralbioactivepeptides.,KRistheabbreviationoflysine,arginine,MSH,melanocyte-stimulatinghormone(促黑激素）,ACTH,adrenocorticotropichormone（促肾上腺皮质激素）,-LT,-lipotropin（脂酸释放激素）,83,(3)Thepolymerizationofsubunits,Someproteinshavequarternarystructurebutareexpressedfromdifferentgenes.,Thetypicalexampleishemoglobin,Therefore,thepolypeptidechainswouldbepolymerizedtoformthequarternarystructureafterthebiosynthesisofpolypeptidechain.,84,Post-translationalprocessing,Polymerizationofsubunits,Oligomer,85,(4)Targettransportationofproteins,Thefinallocationofaproteinresultsfromaprocesscalledproteintargeting.,Thetargetingofproteindependsonthesignalthatapolypeptidecontains.,Variousproteinshavedifferentfinallocationafterreleasedfromribosome.,86,Blood,Proteintargeting,nucleus,mit.,Sidecell,ERandGsystem,cytoplasm,ribosome,87,Thetargetingofnewlyproducingproteins：,88,TheprecursorofsecretaryproteincontainsasignalpeptideattheNterminal,andwillbecutoffaftertransportation.,Signalpeptideisrichinhydrophobicaminoacidsforfacilitatingthetransportation,Secretoryprotein,89,Thesignalpeptideanditsstructure,BasicN-end,Hydrophobiccentre,Cleavagesite,N,Joiningwithsecretoryprotein,ALWRLLPLLALLALWGPDPAAFV.,MMAAGPRTSLLLLAFALLCLPWTQVVGAF.,MKFLVNVALVFMVVYISTITAAP.,Cattlegrowthhormone,Humanpre-insulin,Beevenompeptide,C,90,Questions,1.Thestatement(fromCampbell,)thattheaminoacidsequenceofaproteincontainsalloftheinformationrequiredforthecompletethreedimensionalstructure,A.cannotbefalsifiedbyexperiment;henceitistrivial.B.isstronglysupportedbymanydenaturation-renaturationexperiments.C.iscalledanhypothesissinceitsvalidityissortofdicey.D.iscalledamodelbecauseitcanbediagrammedintextbooks.E.hasessentiallybeenprovenbyrecenttheoreticalwork.,91,SectionFive,Theinterferenceandinhibitionofproteinbiosynthesis,Clinicalcorrelationofproteinbiosynthesis,(1)Moleculardiseases,(2)Effectofantibioticsonproteinbiosynthesis,(3)Otherbioactivematerialsinterferewithproteinbiosynthesis(interferon),93,(2)Effectofantibioticsonproteinbiosynthesis,Someantibioticinhibitorsofproteinbiosynthesis,Antibioticactionstepsaffected,Tetracyclinebindstothe30Ssubunit,inhibitsentryof(四环素族）bindingofaminoacyl-tRNAaminoacyl-tRNA,Streptomycinbindstothe30Ssubunit,