分子生物学（杨洋）第七章 翻译-3

Topic8

Antibiotics(抗生素）andtranslation2026/5/102Antibioticsinhibitcelldivisionbyblockingspecificstepsintranslation—a

powerfultooltofightdiseaseTargetsofmanyantibioticsaretranslationalmachinery2026/5/103OverviewoftheeventsoftranslationTermination

Elongation

Initiation链霉素氯霉素四环素红霉素Moleculartarget:peptidyltransferasereactioncenterof50SsubunitInhibitpeptidyltransferasereactionspecifically氯霉素四环素Moleculartarget:Asiteof30SsubunitInhibitaminoacylated-tRNAbindingtoAsite2026/5/107Inadditiontomedicalbenefits,antibioticscanplayimportantroleinresearchforunderstandingthefunctionoftranslationalmachineryPuromycin(嘌呤霉素)Puromycinresemblestheaminoacylated-tRNA,soitcanbindtotheAsiteofribosome

Puromycin(嘌呤霉素)肽酰嘌呤霉素(peptidyl-puromycin)PuromycinPuromycin(嘌呤霉素)causespolypeptidesynthesistoterminateprematurelyReleaseofpeptidyl-puromycinfromribosomeUsepuromycintostudythepeptidyltransferasereactionandprovethatribosomeisaribozyme(核酶)ExampleI:

Usepuromycintoexplorethetranslationalmechanism:

2026/5/1013Basicconception:whatispeptidyltransferasereaction?

NewpeptidebondPeptidyltransferasereaction(肽酰转移酶反应)N-terminalC-terminalRaisescientificquestionabout

peptidyltransferasereaction(提出科学问题）Whichpartofribosomeisresponsibleforpeptidyltransferasereaction?(50Ssubunitor30Ssubunit?)Nextquestion:rRNAorproteinisresponsibleforpeptidyltransferasereaction?2026/5/1017Howtoanswerthescientificquestion(解决科学问题)EstablishagoodmodelDesigntheelegantexperimentAnalyzetheexperimentalresultsAnswerthescientificquestionStudythepeptidyltransferasereactionWeshouldestablishasystemwhichcandetectpeptidyltransferaseactivityeasilyandinvitro(体外)EstablishagoodmodelPuromycincanbeusedtodetectthepeptidyltransferasereactioninvitroModel:poly(U)-ribosome-poly(Phe)(14C-labeled)UUU--Phepoly(U)—poly(Phe)放射性标记(14C-labeled)Releaseofpeptidyl-puromycinfromribosomeDetectionofthe14C-labeledreleasedpeptidyl-puromycincanreflectthepeptidyltransferase

activityFirst

question:Whichpartofribosomeisresponsibleforpeptidyltransferasereaction?(50Ssubunitor30Ssubunit?)Localizationofpeptidyltransferase[14C]-poly(Phe)Remove30Ssubuint50Ssubunit/poly(Phe)-tRNA

Detectionofthe14C-labeledreleasedpeptidyl-puromycincanreflectthepeptidyltransferase

activity50Ssubunitofribosomealonecanhavepeptidyltransferaseactivity蔗糖密度梯度离心[14C]Howcanwedistinguishthereleased14C-poly(Phe)-puro

fromthe14C-poly(Phe)-ribosome?

[14C]Firstquestion:Answer:50SsubunithaspeptidyltransferaseactivityLocalizationofpeptidyltransferaseto50Ssubunit

Whichpartofribosomeisresponsibleforpeptidyltransferasereaction?(50Ssubunitor30Ssubunit?)Nextquestion:Whichcomponentof50Ssubunithasthepeptidyltransferaseactivity?(proteinorrRNA?)

Beforetheribozymeconceptwasputforward,theanswerwasobvious:onlyproteincanhavepeptidyltransferaseactivity!1980s,Cechputforwardtheribozyme(核酶）concept:RNAcanalsohavecatalysefunctionasenzyme

Hypothesis:23SrRNAinthe50Ssubunitcancatalyzethepeptidyltransferasereaction?Howcanweprovethishypothesis?ThepeptidyltransferasereactioncenterislocatedinthelargesubunitofribosomeandcomposedalmostentirelyofrRNAHypothesis:Peptidyltransferaseisaribozyme(核酶),whichiscomposedofRNAWhatistheexperimentalproof?Howcanweprovethishypothesis?Usepuromycintostudythepeptidyltransferasereactionandprovidebiochemicalevidenceforribosomebeingaribozyme(核酶)研究论文（原始研究工作）的学习ResearchstrategyRemovealltheproteinsfrom50SsubunitLeaveonlytherRNADetectwhetherrRNAalonecanhavepeptidyltransferaseactivity?Reagentwhichcanremoveproteins:phenol(苯酚),SDS,proteinaseK(PK,蛋白酶K)2026/5/1031Question:Howcanwedetectpeptidyltransferaseactivitysimplyinvitro(体外)?PuromycincanbeusedtodetectthepeptidyltransferasereactioninvitroAUG50S[35S]fMet-tRNAfMetPsiteAsitepuromycinaddingAUG50SfMet-tRNAfMetPsiteAsitepuropeptidyltransferasereactionDetectionofreleased[35S]fMet-puromycincomplexpuro[35S]fMetModel:50Ssubunit--AUG--[35S]fMet-tRNAfMet--puromycinPeptidyltransferaseactivitycanbemonitoredby[35S]fMet-puromycincomplex---片段反应Reagentwhichcanremoveproteins:phenol(苯酚),SDS,proteinaseK(PK,蛋白酶K)Treat50SsubunittoremoveproteinsDetectthepeptidyltransferasereaction[35S]fMet-puromycincomplexcanreflectthepeptidyltransferaseactivitylane1:E.coli70Sribosome/[35S]fMet-tRNA/puromycin

peptidyltransferaseactivity(+)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KNextquestion:Whythephenol

candisruptthepeptidyltransferase?Whytheothertworeagentswhichcanalsoremoveproteins(PKandSDS)

cannotdisruptthepeptidyltransferase?PhenolmorevigorousthanPKandSDS?Hypothesis:Phenolperhapsdisruptedthehigh-orderstructureofRNAwhichwasessentialforthepeptidyltransferaseactivityDesignthefollowingexperimenttoprovethishypothesisquestionhypothesisExperimenttesthypothesisanswerHowtotestthishypothesis?Strategy:findarRNAwhichismorestablethanthatofE.coliandnotsensitivetophenolRNAcankeepitsnativehigh-orderstructure

evenafterphenolextraction50SsubunitfromthermophilicbacteriumThermusaquaticus(嗜热菌）rRNAfromthermophilicbacteriummightbemoresturdythanthatofE.coliRNAfromthermophilicbacteriumcankeepitsnativehigh-orderstructureevenafterphenolextractionribosomeSDSPKphenolpeptidyltransferase+_++++[35S]fMet-puromycincomplexlane4: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+phenolPhenolperhapsdisruptedthehigh-orderstructureofRNAwhichwasessentialforthepeptidyltransferaseactivityRNAfromThermusaquaticuscankeepitsnativehigh-orderstructureevenafterphenolextractionSothepeptidyltransferaseactivitywasresistanttophenolWhythephenol

candisruptthepeptidyltransferase?

2026/5/1041rRNAalonecanhavepeptidyltransferaseactivity,notprotein2026/5/1042NEXTquestion:Doesthe[35S]fMet-puromycincomplexreallyrepresentthepeptidyltransferaseactivity?DoesRNAreallyhavethepeptidyltransferaseactivity?Chloramphenicol(氯霉素)Carbomycin(碳霉素)peptidyltransferaseinhibitor（肽酰转移酶抑制剂）RNaseT1DegradeRNAFurtherexperimentalproof:[35S]fMet/puromycinT.aquaticus50Sribosome/[35S]fMet-tRNA/puromycincontrol1234lane2：addchloramphenicol(氯霉素)lane3：addcarbomycin(碳霉素)lane4：addRNaseT15lane6：addchloramphenicol(氯霉素)andSDS-PK-phenol678lane7：addcarbomycin(碳霉素)andSDS-PK-phenollane8：addRNaseT1andSDS-PK-phenolErythromycin(红霉素):notaffectpeptidyltransferase

Anisomycin(茴香霉素):inhibit

peptidyltransferaseineukaryoticribosome,butnotinbacterialribosome

TreatT.aquaticus50SribosomewithErythromycinandAnisomycin

Whatwillhappen?ribosomeErythromycinpeptidyltransferaseAnisomycinChloramphenicol+++------++-T50ST50S:Thermusaquaticus50Sribosomesubunit红霉素茴香霉素氯霉素Questionaboutthepeptidyltransferasereaction:Whichpartofribosomeisresponsibleforpeptidyltransferasereaction?(50Ssubunitor30Ssubunit?