分子生物学(杨洋)第七章翻译课件

2020/5/9,1,Outline,Topics1-4:Fourcomponentsoftranslationmachinery.T1-mRNA;T2-tRNA;T3-AttachmentofaminoacidstotRNA(aminoacyl-tRNAsynthetases);T4-ribosomeTopic5-7:Translationprocess.T5-initiation;T6-elongation;T7-termination.,2020/5/9,2,TranslationprocessT5:InitiationoftranslationT6:ElongationoftranslationT7:terminationoftranslation,2020/5/9,3,Overviewoftheeventsoftranslation,Termination,Elongation,Initiation,Topic5:Initiationoftranslation,Threeeventsmustoccur:ribosomemustberecruitedtomRNA2.achargedtRNAmustbeplacedintothePsiteofribosome3.theribosomemustbepreciselypositionedoverthestartcodon,2020/5/9,6,startcodon,One-baseshiftwillresultinthesynthesisofcompletelyunrelatedpolypeptide,ThedifferentstructureofprokaryoticandeukaryoticmRNAresultsindistinctlydifferentmeansoftranslationalinitiation,2020/5/9,8,5-1prokaryoticmRNAsarerecruitedtothesmallsubunitbybasepairingtorRNA,mRNA,mRNA/smallsubunitcomplex,2020/5/9,9,5-2AspecializedtRNAchargedwithmodifiedmethioninebindsdirectlytoprokaryoticsmallsubunit,InitiatortRNA:fMet-tRNA(甲酰甲硫氨酸),fMet-tRNAfMet,2020/5/9,10,modifiedmethionine,(甲酰甲硫氨酸),5-3Threeinitiationfactors(IF)directtheassemblyofaninitiationcomplexthatcontainsmRNAandinitiatortRNA,Translationinitiationfactors(IF):IF1:preventtRNAfrombindingtotheAsiteinthesmallsubunitIF2:aGTPasethatinteractswithsmallsubunit,IF1andinitiatortRNA(fMet-tRNAfMet);IF2canfacilitatetheassociationoffMet-tRNAfMetwiththesmallsubunitIF3:bindstothesmallsubunitandblocksitfromreassociatingwithlargesubunit,helpstodissociatethe70Sribosomeintoitslargeandsmallsubunit,2020/5/9,12,ThreebindingsitefortRNAs,Asite:tobindtheaminoacylated-tRNAP-site:tobindthepeptidyl-tRNAE-site:tobindtheunchargedtRNA,2020/5/9,13,EachoftheinitiationfactorsbindsatornearoneofthethreetRNA-bindingsitesonthesmallsubunit,IF3,30Ssmallsubunit,50Slargesubunit,IF1,IF2,fMet-tRNAfMet,50Slargesubunit,IF3,70Sinitiationcomplex,IF2-GTP,IF2-GDP,IF1,50Slargesubunit,remove,5-4EukaryoticribosomearerecruitedtomRNAbythe5-Cap,Similarity:bothuseastartcodonandinitiatortRNAbothuseinitiationfactorstoformmRNA/smallsubunitcomplexbeforeadditionoflargesubunit,Comparisonbetweenprokaryoticandeukaryotictranslationalinitiation,Difference:themethodofrecognizingthemRNAandstartcodonisfundamentallydistinct,2020/5/9,16,Once,Kozaksequence,EukaryoticmRNAusesamethylatedcaptorecruittheribosome.Oncebound,theribosomescansthemRNAina5-3directiontofindtheAUGstartcodon-scanning(扫描)Kozaksequencecanincreasethetranslationefficiency,2020/5/9,17,40Ssmallsubunit,Met-tRNAMet,Hairpin:blockthebindingbetweenmRNAandribosome,elF4BactivatestheRNAhelicase(RNA解旋酶)inoneoftheelF4Fsubunits,mRNA/smallsubunitcomplex,Unwindthehairpinstructure,2020/5/9,18,5-5Thestartcodonisfoundbyscanningdownstreamfrom5-endofmRNA,2020/5/9,19,Identificationofthestartcodonbysmallribosomesubunit,Met-tRNAMetisplacedinthePsiteofthe80Sinitiationcomplex,scanning,2020/5/9,20,Scientificquestion(提出科学问题),HowtheeukaryoticribosomerecognizethestartcodonAUG?WhatisthefunctionofinitiatortRNA-Met-tRNAMetinthetranslationinitiationofeukaryoticcell?,研究论文（原始研究工作）的学习,2020/5/9,22,设计实验,实验数据分析,得出结论,提出科学问题,分析科学问题,解决科学问题,2020/5/9,23,tRNAiMetfunctionsindirectingthescanningribosometothestartsiteoftranslation.Science,Vol242,Issue4875,93-97,Keywords:TranslationinitiationRibosomeStartcodonScanningmodelMet-tRNAMet,RibosomebindingsitesoftheprokaryotictypearenotpresentineukaryoticmRNAEukaryoticscanningmodel:The40SribosomalsubunitinassociationwithinitiatortRNA(Met-tRNAMet)andinitiationfactorsbindsthe5-capofthemRNAmigratesina5to3direction,“scanning”foratranslationalstartsite,AUG,Eukaryotictranslationinitiation,initiatortRNA(Met-tRNAMet),40Ssubunit,尚未解决的科学问题：,TheprecisemechanismbywhichthescanningribosomerecognizesthefirstAUGcodonhasnotbeendiscoveredWhatistheroleofinitiatortRNA(Met-tRNAMet)inthetranslationinitiationprocess?,2020/5/9,27,Howtoanswerthescientificquestion(解决科学问题),Establishagoodmodel,Designtheelegantexperiment,Analyzetheexperimentalresults,Answerthescientificquestion,Model:yeast,Saccharomycescerevisiae(酿酒酵母）-modelorganismofeukaryotes,Hisgene(编码合成组氨酸的基因）:necessarygeneforyeastgrowthonthebasicmediumlackinghistidine,Cangrowonthebasicmediumlackinghistidine,Phenotype:His+,AGG,5,Hisgene,Mutantyeastcell,Cannotgrowonthebasicmediumlackinghistidine,Phenotype:His-,Asimplegrowthtest:reflecttranslationofhisgene,AGG,5,Hisgene,UUG,5,Hisgene,AUC,5,Hisgene,AAG,5,Hisgene,ACG,5,Hisgene,AUG,5,Hisgene,Wild-type,Mutant-1,Mutant-2,Mutant-3,Mutant-4,Mutant-5,His+,His-,His-,His-,His-,His-,genotype,phenotype,UAC,Met,Met-tRNAMet(initiatortRNA),G,Phenotype:His+,UAC,G,Met,Met-tRNAMet(initiatortRNA),3,5,mutation,UCC,3,5,Met,mutation,tRNAgenewasmutated,MutatedtRNA,High-copyplasmidOver-expressionofmutatedtRNAgene,AGG,5,Hisgene,Mutantyeastcell,导入,UUG,AUC,AAG,ACG,growthtestonthebasicmediumlackinghistidine,Met,growth,Mutatedstartcodon,High-copyplasmidOver-expressionofmutatedtRNAgene,ACG,5,Hisgene,Mutantyeastcell,导入,Cannotgrowonthebasicmediumlackinghistidine,mutatedtRNA,Met,High-copyplasmidOver-expressionofmutatedtRNAgene,AGG,5,Hisgene,Mutantyeastcell,导入,Cangrowonthebasicmediumlackinghistidine,mutatedtRNA,Met,AGG,5,Hisgene,Mutantyeastcell,Cannotgrowonthebasicmediumlackinghistidine,Wild-typetRNA,Met,AGG,5,Hisgene,Mutantyeastcell-2,AGG,+1,-28,Anotherexperiment:,AGG,5,Hisgene,Mutantyeastcell-1,ACG,+1,-28,High-copyplasmidOver-expressionofmutatedtRNAgene,导入,mutatedtRNA,AGG,5,Hisgene,Mutantyeastcell-2,AGG,+1,-28,growthtestonthebasicmediumlackinghistidine,Met,High-copyplasmidOver-expressionofmutatedtRNAgene,导入,mutatedtRNA,AGG,5,Hisgene,Mutantyeastcell-1,ACG,+1,-28,growthtestonthebasicmediumlackinghistidine,Met,+,+,growth,His-,His+,TheupstreamAGGoutofthereadingframeofhisgeneservesasasiteofinitiation,hisgene,hisgene,ACG,-28,ThemutanttRNAalsodirectedtheribosometoinitiateatanAGGplacedintheupstreamregionofthehisgene,High-copyplasmidOver-expressionofmutatedtRNAgene,导入,mutatedtRNA,AGG,5,Hisgene,Mutantyeastcell-2,AGG,+1,-28,growthonthebasicmediumlackinghistidinewasinhibited,Provethescanningmodel,SD:GUGUG,Humangrowthhormonegene,Producemuchhumangrowthhormoneprotein,mutatedtypeE.colicell(16srRNA:CACAC),Ananticodon:codoninteractionbetweentRNAMetandthefirstAUGinthemRNAisimportantforribosomalrecognitionofthetranslationalstartsiteduringthescanningprocess,5-6TranslationinitiationfactorsholdeukaryoticmRNAsincircles,ItcanexplainhowthemRNApoly-Atailcontributestothetranslationefficiency.,Fig14-29,elF4F,2020/5/9,46,翻译起始的小结:,SimilarityanddifferencebetweenprokaryoticandeukaryotictranslationalinitiationFunctionoftranslationinitiationfactors:IF1,IF2,IF3eIF1A,eIF3,eIF2,eIF4F,eIF4B,Topic6Elongationoftranslation,Threeimportanteventsintheelongationoftranslation,进位,易位,肽键形成,Elongationfactor:EF-Tuescortaminoacylated-tRNAtotheAsiteoftheribosome,Elongationfactor:EF-GstimulatesthetranslocationoftRNA,EF-G-GDP,肽键形成(肽转移酶反应）,2020/5/9,52,Ribosomeisaribozyme(核酶):PeptidyltransferasereactioniscatalyzedbyRNA,specially23srRNA,Catalysisrequiresdistanceinthe1-3,andonlyRNAresiduesarepresent18fromtheactivesite.,RNAsurroundthepeptidyltransferasecenterofthelargeribosomesubunit,2020/5/9,53,翻译延伸小结:,ThreeimportanteventsintheelongationoftranslationFunctionoftranslationelongationfactors:EF-Tu,EF-GPeptidyltransferasereaction,Ribosomeisaribozyme(核酶),Topic7:terminationoftranslation,2020/5/9,55,Overviewoftheeventsoftranslation,Termination,Elongation,Initiation,2020/5/9,56,ThestopcodonUAG,UGA,orUAA(5-3)Functions:1.DefinestheendofORF2.Signalterminationofpolypeptidesynthesis,2020/5/9,57,Question:,Howtheribosomecanrecognizethestopcodon?Whatisthemechanism?,UAG,peptide,Postulation:Chain-terminatingtRNAforrecognizingthestopcodon?Releasefactor(RF,释放因子）istRNA?,Chain-terminatingtRNA,?,MarioCapecchiresearchworkdiscoveryofreleasefactor(RF,释放因子）,model:E.coliribosomeandphageR17mRNAinvitro(体外）translationsystem,Theexperimentforfindingthereleasefactor,(+RNA酶处理）,不加苏氨酸,加14C苏氨酸,(+蛋白酶处理）,加入核糖体上清液组分(ContainingRF)（whatisRF?),五肽,14C标记的六肽,能够释放多肽链,不能够释放多肽链,Discovery:,Releasefactor(RF,释放因子）isprotein,notRNAProteinscalledreleasefactors(RFs)recognizethestopcodonandactivatethehydrolysisofthepolypeptidefrompeptidyl-tRNA,Twoclassesofreleasefactors,ClassI:recognizethestopcodonandtriggerhydrolysisofthepolypeptidefrompeptidyl-tRNAProkaryotes:RF1-UAG,UAARF2-UGA,UAAEukaryotes:eRF1-UAG,UGA,UAAClassII:stimulatethedissociationoftheclassIfactorsfromtheribosomeafterreleaseofpolypeptideProkaryotes:RF3Eukaryotes:eRF3GTP-bindingprotein,2020/5/9,63,NirenbergresearchworkmoredirectproveforthefunctionofRF,Prokaryotes:RF1-UAG,UAARF2-UGA,UAA,Ribosome-AUG-3HfMet-tRNAfMetcomplex,3HfMet,3HfMet,3HfMet,FurtherquestionaboutClassIreleasefactor,Howdoreleasefactorsrecognizethestopcodon?Whatisthemechanismofthisrecognition?,Howtheribosomecanrecognizethestopcodon?,Proteinscalledreleasefactors(RFs)recognizethestopcodonandactivatethehydrolysisofthepolypeptidefrompeptidyl-tRNA,Protein(RF)-RNA(mRNA)interactionHypothesis:RFmimicsthestructureandfunctionoftRNARFcancompetewithtRNAforbindingtotheribosomeandrecognizingcodonsastRNA,UAG,peptide,Postulation:Chain-terminatingtRNAforrecognizingthestopcodon?Releasefactor(RF,释放因子）istRNA?,Chain-terminatingtRNA,?,Thesiteofattachmentofaminoacid,Athree-nucleotide-longsequenceresponsibleforrecognizingthecodonbybasepairingwithmRNA,cloverleafstructureoftRNA,Peptideanticodon(肽反密码子):three-aminoacidssequenceinRFwhichcanrecognizeandinteractwiththestopcodon,Cell,2000,101:349-352,UAG,UAA,UGA,UAA,Peptideanticodon(肽反密码子):three-aminoacidssequenceinRFwhichcanrecognizeandinteractwiththestopcodonDeterminethespecificityofRF1andRF2forrecognizingthestopcodon,GGQsequence(Gly-Gly-Gln):aregionofclassIreleasefactorwhichcontributestopolypeptidereleasefromtRNA,AnotherimportantregioninRF:,Qu