分子生物学(杨洋)第七章翻译

Topic8Antibiotics 抗生素 andtranslation 2020 4 20 2 Antibioticsinhibitcelldivisionbyblockingspecificstepsintranslation apowerfultooltofightdiseaseTargetsofmanyantibioticsaretranslationalmachinery 2020 4 20 3 Overviewoftheeventsoftranslation Termination Elongation Initiation 链霉素 氯霉素 四环素 红霉素 Moleculartarget peptidyltransferasereactioncenterof50Ssubunit Inhibitpeptidyltransferasereactionspecifically 氯霉素 四环素 Moleculartarget Asiteof30Ssubunit Inhibitaminoacylated tRNAbindingtoAsite 2020 4 20 7 Inadditiontomedicalbenefits antibioticscanplayimportantroleinresearchforunderstandingthefunctionoftranslationalmachinery Puromycin 嘌呤霉素 Puromycinresemblestheaminoacylated tRNA soitcanbindtotheAsiteofribosome Puromycin 嘌呤霉素 肽酰嘌呤霉素 peptidyl puromycin Puromycin Puromycin 嘌呤霉素 causespolypeptidesynthesistoterminateprematurely Releaseofpeptidyl puromycinfromribosome Usepuromycintostudythepeptidyltransferasereactionandprovethatribosomeisaribozyme 核酶 ExampleI Usepuromycintoexplorethetranslationalmechanism 2020 4 20 13 Basicconception whatispeptidyltransferasereaction Newpeptidebond Peptidyltransferasereaction 肽酰转移酶反应 N terminal C terminal Raisescientificquestionaboutpeptidyltransferasereaction 提出科学问题 Whichpartofribosomeisresponsibleforpeptidyltransferasereaction 50Ssubunitor30Ssubunit Nextquestion rRNAorproteinisresponsibleforpeptidyltransferasereaction 2020 4 20 17 Howtoanswerthescientificquestion 解决科学问题 Establishagoodmodel Designtheelegantexperiment Analyzetheexperimentalresults Answerthescientificquestion StudythepeptidyltransferasereactionWeshouldestablishasystemwhichcandetectpeptidyltransferaseactivityeasilyandinvitro 体外 Establishagoodmodel Puromycincanbeusedtodetectthepeptidyltransferasereactioninvitro Model poly U ribosome poly Phe 14C labeled UUU Phe poly U poly Phe 放射性标记 14C labeled Releaseofpeptidyl puromycinfromribosome Detectionofthe14C labeledreleasedpeptidyl puromycincanreflectthepeptidyltransferaseactivity Firstquestion Whichpartofribosomeisresponsibleforpeptidyltransferasereaction 50Ssubunitor30Ssubunit Localizationofpeptidyltransferase 14C poly Phe Remove30Ssubuint 50Ssubunit poly Phe tRNA Detectionofthe14C labeledreleasedpeptidyl puromycincanreflectthepeptidyltransferaseactivity 50Ssubunitofribosomealonecanhavepeptidyltransferaseactivity 蔗糖密度梯度离心 14C Howcanwedistinguishthereleased14C poly Phe purofromthe14C poly Phe ribosome 14C Firstquestion Answer 50SsubunithaspeptidyltransferaseactivityLocalizationofpeptidyltransferaseto50Ssubunit Whichpartofribosomeisresponsibleforpeptidyltransferasereaction 50Ssubunitor30Ssubunit Nextquestion Whichcomponentof50Ssubunithasthepeptidyltransferaseactivity proteinorrRNA Beforetheribozymeconceptwasputforward theanswerwasobvious onlyproteincanhavepeptidyltransferaseactivity 1980s Cechputforwardtheribozyme 核酶 concept RNAcanalsohavecatalysefunctionasenzyme Hypothesis 23SrRNAinthe50Ssubunitcancatalyzethepeptidyltransferasereaction Howcanweprovethishypothesis ThepeptidyltransferasereactioncenterislocatedinthelargesubunitofribosomeandcomposedalmostentirelyofrRNA Hypothesis Peptidyltransferaseisaribozyme 核酶 whichiscomposedofRNA Whatistheexperimentalproof Howcanweprovethishypothesis Usepuromycintostudythepeptidyltransferasereactionandprovidebiochemicalevidenceforribosomebeingaribozyme 核酶 研究论文 原始研究工作 的学习 Researchstrategy Removealltheproteinsfrom50Ssubunit LeaveonlytherRNA DetectwhetherrRNAalonecanhavepeptidyltransferaseactivity Reagentwhichcanremoveproteins phenol 苯酚 SDS proteinaseK PK 蛋白酶K 2020 4 20 31 Question Howcanwedetectpeptidyltransferaseactivitysimplyinvitro 体外 Puromycincanbeusedtodetectthepeptidyltransferasereactioninvitro AUG 50S 35S fMet tRNAfMet Psite Asite puromycin adding AUG 50S fMet tRNAfMet Psite Asite puro peptidyltransferasereaction Detectionofreleased 35S fMet puromycincomplex puro 35S fMet Model 50Ssubunit AUG 35S fMet tRNAfMet puromycin Peptidyltransferaseactivitycanbemonitoredby 35S fMet puromycincomplex 片段反应 Reagentwhichcanremoveproteins phenol 苯酚 SDS proteinaseK PK 蛋白酶K Treat50Ssubunittoremoveproteins Detectthepeptidyltransferasereaction 35S fMet puromycincomplexcanreflectthepeptidyltransferaseactivity lane1 E coli70Sribosome 35S fMet tRNA puromycinpeptidyltransferaseactivity lane2 E coli70Sribosome 35S fMet tRNA puromycin SDS PKpeptidyltransferaseactivity lane3 E coli50Sribosome 35S fMet tRNA puromycin SDS PKpeptidyltransferaseactivity lane4 E coli70Sribosome 35S fMet tRNA puromycin phenolpeptidyltransferaseactivity PK 蛋白酶K Nextquestion Whythephenolcandisruptthepeptidyltransferase Whytheothertworeagentswhichcanalsoremoveproteins PKandSDS cannotdisruptthepeptidyltransferase PhenolmorevigorousthanPKandSDS Hypothesis Phenolperhapsdisruptedthehigh orderstructureofRNAwhichwasessentialforthepeptidyltransferaseactivity Designthefollowingexperimenttoprovethishypothesis question hypothesis Experiment testhypothesis answer Howtotestthishypothesis Strategy findarRNAwhichismorestablethanthatofE coliandnotsensitivetophenol RNAcankeepitsnativehigh orderstructureevenafterphenolextraction 50SsubunitfromthermophilicbacteriumThermusaquaticus 嗜热菌 rRNAfromthermophilicbacteriummightbemoresturdythanthatofE coli RNAfromthermophilicbacteriumcankeepitsnativehigh orderstructureevenafterphenolextraction ribosome SDS PK phenol peptidyltransferase 35S fMet puromycincomplex lane4 E coli70Sribosome 35S fMet tRNA puromycin phenolpeptidyltransferaseactivity lane8 T aquaticus50S 35S fMet tRNA puromycin SDS PK phenolpeptidyltransferaseactivity lane5 Thermusaquaticus50Sribosome 35S fMet tRNA puromycinpeptidyltransferaseactivity lane6 Thermusaquaticus50Sribosome 35S fMet tRNA puromycin SDSpeptidyltransferaseactivity lane7 T aquaticus50Sribosome 35S fMet tRNA puromycin SDS PKpeptidyltransferaseactivity Lane11 Thermusaquaticus70Sribosome 35S fMet tRNA puromycin phenol Phenolperhapsdisruptedthehigh orderstructureofRNAwhichwasessentialforthepeptidyltransferaseactivity RNAfromThermusaquaticuscankeepitsnativehigh orderstructureevenafterphenolextraction Sothepeptidyltransferaseactivitywasresistanttophenol Whythephenolcandisruptthepeptidyltransferase 2020 4 20 41 rRNAalonecanhavepeptidyltransferaseactivity notprotein 2020 4 20 42 NEXTquestion Doesthe 35S fMet puromycincomplexreallyrepresentthepeptidyltransferaseactivity DoesRNAreallyhavethepeptidyltransferaseactivity Chloramphenicol 氯霉素 Carbomycin 碳霉素 peptidyltransferaseinhibitor 肽酰转移酶抑制剂 RNaseT1 DegradeRNA Furtherexperimentalproof 35S fMet puromycin T aquaticus50Sribosome 35S fMet tRNA puromycin control 1 2 3 4 lane2 addchloramphenicol 氯霉素 lane3 addcarbomycin 碳霉素 lane4 addRNaseT1 5 lane6 addchloramphenicol 氯霉素 andSDS PK phenol 6 7 8 lane7 addcarbomycin 碳霉素 andSDS PK phenol lane8 addRNaseT1andSDS PK phenol Erythromycin 红霉素 notaffectpeptidyltransferase Anisomycin 茴香霉素 inhibitpeptidyltransferaseineukaryoticribosome butnotinbacterialribosome TreatT aquaticus50SribosomewithErythromycinandAnisomycin Whatwillhappen ribosome Erythromycin peptidyltransferase Anisomycin Chloramphenicol T50S T50S Thermusaquaticus50Sribosomesubunit 红霉素 茴香霉素 氯霉素 Questionaboutthepeptidyltransferasereaction Whichpartofribosomeisresponsibleforpeptidyltransferasereaction 50Ssubunitor30Ssubunit rRNAorprotein