分子生物学（杨洋）第七章 翻译-3

Topic8Antibiotics(抗生素）andtranslation5/7/20261Antibioticsinhibitcelldivisionbyblockingspecificstepsintranslation—a

powerfultooltofightdiseaseTargetsofmanyantibioticsaretranslationalmachinery5/7/20262OverviewoftheeventsoftranslationTermination

Elongation

Initiation5/7/20263链霉素氯霉素四环素红霉素5/7/20264Moleculartarget:peptidyl

transferasereactioncenterof50SsubunitInhibitpeptidyl

transferasereactionspecifically氯霉素5/7/20265四环素Moleculartarget:Asiteof30SsubunitInhibitaminoacylated-tRNAbindingtoAsite5/7/20266Inadditiontomedicalbenefits,antibioticscanplayimportantroleinresearchforunderstandingthefunctionoftranslationalmachinery5/7/20267Puromycin(嘌呤霉素)Puromycinresemblestheaminoacylated-tRNA,soitcanbindtotheAsiteofribosome

5/7/202685/7/20269Puromycin(嘌呤霉素)肽酰嘌呤霉素(peptidyl-puromycin)Puromycin5/7/202610Puromycin(嘌呤霉素)causespolypeptidesynthesistoterminateprematurelyReleaseofpeptidyl-puromycinfromribosome5/7/202611Usepuromycintostudythepeptidyl

transferasereactionandprovethatribosomeisaribozyme(核酶)ExampleI:

Usepuromycintoexplorethetranslationalmechanism:

5/7/202612Basicconception:whatispeptidyl

transferasereaction?

5/7/202613NewpeptidebondPeptidyl

transferasereaction(肽酰转移酶反应)N-terminalC-terminal5/7/202614Raisescientificquestionabout

peptidyl

transferasereaction(提出科学问题）Whichpartofribosomeisresponsibleforpeptidyl

transferasereaction?(50Ssubunitor30Ssubunit?)5/7/202615Nextquestion:rRNAorproteinisresponsibleforpeptidyl

transferasereaction?5/7/202616Howtoanswerthescientificquestion(解决科学问题)EstablishagoodmodelDesigntheelegantexperimentAnalyzetheexperimentalresultsAnswerthescientificquestion5/7/202617Studythepeptidyl

transferasereactionWeshouldestablishasystemwhichcandetectpeptidyl

transferaseactivityeasilyandinvitro(体外)EstablishagoodmodelPuromycincanbeusedtodetectthepeptidyl

transferase

reactioninvitro5/7/202618Model:poly(U)-ribosome-poly(Phe)(14C-labeled)UUU--Phepoly(U)—poly(Phe)放射性标记(14C-labeled)Releaseofpeptidyl-puromycinfromribosomeDetectionofthe14C-labeledreleasedpeptidyl-puromycincanreflectthepeptidyl

transferase

activity5/7/202619First

question:Whichpartofribosomeisresponsibleforpeptidyl

transferasereaction?(50Ssubunitor30Ssubunit?)Localizationofpeptidyl

transferase5/7/202620[14C]-poly(Phe)Remove30Ssubuint50Ssubunit/poly(Phe)-tRNA

Detectionofthe14C-labeledreleasedpeptidyl-puromycincanreflectthepeptidyl

transferase

activity50Ssubunitofribosomealonecanhavepeptidyl

transferaseactivity5/7/202621蔗糖密度梯度离心[14C]Howcanwedistinguishthereleased14C-poly(Phe)-puro

fromthe14C-poly(Phe)-ribosome?

[14C]5/7/2026225/7/202623Firstquestion:Answer:50Ssubunithaspeptidyl

transferaseactivityLocalizationofpeptidyl

transferaseto50Ssubunit

Whichpartofribosomeisresponsibleforpeptidyl

transferasereaction?(50Ssubunitor30Ssubunit?)5/7/202624Nextquestion:Whichcomponentof50Ssubunithasthepeptidyl

transferaseactivity?(proteinorrRNA?)

Beforetheribozymeconceptwasputforward,theanswerwasobvious:onlyproteincanhavepeptidyl

transferaseactivity!5/7/2026251980s,Cechputforwardtheribozyme

(核酶）concept:RNAcanalsohavecatalysefunctionasenzyme

Hypothesis:23SrRNAinthe50Ssubunitcancatalyzethepeptidyl

transferasereaction?Howcanweprovethishypothesis?5/7/202626Thepeptidyl

transferasereactioncenterislocatedinthelargesubunitofribosomeandcomposedalmostentirelyofrRNAHypothesis:Peptidyl

transferaseisaribozyme(核酶),whichiscomposedofRNAWhatistheexperimentalproof?Howcanweprovethishypothesis?Usepuromycintostudythepeptidyl

transferasereactionandprovidebiochemicalevidenceforribosomebeingaribozyme(核酶)5/7/202627研究论文（原始研究工作）的学习5/7/2026285/7/202629ResearchstrategyRemovealltheproteinsfrom50SsubunitLeaveonlytherRNADetectwhetherrRNAalonecanhavepeptidyl

transferaseactivity?Reagentwhichcanremoveproteins:phenol(苯酚),SDS,proteinaseK(PK,蛋白酶K)5/7/202630Question:Howcanwedetectpeptidyl

transferaseactivitysimplyinvitro(体外)?Puromycincanbeusedtodetectthepeptidyl

transferase

reactioninvitro5/7/202631AUG50S[35S]fMet-tRNAfMetPsiteAsitepuromycinaddingAUG50SfMet-tRNAfMetPsiteAsitepuropeptidyl

transferasereactionDetectionofreleased[35S]fMet-puromycincomplexpuro[35S]fMetModel:50Ssubunit--AUG--[35S]fMet-tRNAfMet--puromycinPeptidyl

transferaseactivitycanbemonitoredby[35S]fMet-puromycincomplex---片段反应5/7/202632Reagentwhichcanremoveproteins:phenol(苯酚),SDS,proteinaseK(PK,蛋白酶K)Treat50SsubunittoremoveproteinsDetectthepeptidyl

transferasereaction5/7/202633[35S]fMet-puromycincomplexcanreflectthepeptidyl

transferaseactivitylane1:E.coli70Sribosome/[35S]fMet-tRNA/puromycin

peptidyl

transferaseactivity(+)lane2:E.coli70Sribosome/[35S]fMet-tRNA/puromycin+SDS+PK

peptidyl

transferaseactivity(+)lane3:E.coli50Sribosome/[35S]fMet-tRNA/puromycin+SDS+PK

peptidyl

transferaseactivity(+)lane4:E.coli70Sribosome/[35S]fMet-tRNA/puromycin+phenol

peptidyl

transferaseactivity(-)

PK:蛋白酶K5/7/202634Nextquestion:Whythephenol

candisruptthepeptidyl

transferase?Whytheothertworeagentswhichcanalsoremoveproteins(PKandSDS)

cannotdisruptthepeptidyl

transferase?PhenolmorevigorousthanPKandSDS?5/7/202635Hypothesis:Phenolperhapsdisruptedthehigh-orderstructureofRNAwhichwasessentialforthepeptidyl

transferaseactivityDesignthefollowingexperimenttoprovethishypothesisquestionhypothesisExperimenttesthypothesisanswer5/7/202636Howtotestthishypothesis?Strategy:findarRNAwhichismorestablethanthatofE.coliandnotsensitivetophenolRNAcankeepitsnativehigh-orderstructure

evenafterphenolextraction5/7/20263750SsubunitfromthermophilicbacteriumThermus

aquaticus

(嗜热菌）rRNAfromthermophilicbacteriummightbemoresturdythanthatofE.coliRNAfromthermophilicbacteriumcankeepitsnativehigh-orderstructureevenafterphenolextraction5/7/202638ribosomeSDSPKphenolpeptidyl

transferase

+_++++[35S]fMet-puromycincomplexlane4:E.coli70Sribosome/[35S]fMet-tRNA/puromycin+phenol

peptidyl

transferaseactivity(-)lane8:

T.aquaticus50S/[35S]fMet-tRNA/puromycin+SDS+PK+phenol

peptidyl

transferaseactivity(+)lane5:Thermus

aquaticus50Sribosome/[35S]fMet-tRNA/puromycin

peptidyl

transferaseactivity(+)lane6:Thermus

aquaticus50Sribosome/[35S]fMet-tRNA/puromycin+SDS

peptidyl

transferaseactivity(+)lane7:T.aquaticus50Sribosome/[35S]fMet-tRNA/puromycin+SDS+PK

peptidyl

transferaseactivity(+)

Lane11:Thermus

aquaticus70Sribosome/[35S]fMet-tRNA/puromycin+phenol5/7/202639Phenolperhapsdisruptedthehigh-orderstructureofRNAwhichwasessentialforthepeptidyl

transferaseactivityRNAfromThermus

aquaticuscankeepitsnativehigh-orderstructureevenafterphenolextractionSothepeptidyl

transferaseactivitywasresistanttophenolWhythephenol

candisruptthepeptidyl

transferase?

5/7/202640rRNAalonecanhavepeptidyl

transferaseactivity,notprotein5/7/202641NEXTquestion:Doesthe[35S]fMet-puromycincomplexreallyrepresentthepeptidyl

transferaseactivity?DoesRNAreallyhavethepeptidyl

transferaseactivity?5/7/202642Chloramphenicol(氯霉素)Carbomycin(碳霉素)peptidyl

transferaseinhibitor（肽酰转移酶抑制剂）RNaseT1DegradeRNAFurtherexperimentalproof:5/7/202643[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-phenol5/7/202644Erythromycin(红霉素):notaffectpeptidyl

transferase

Anisomycin(茴香霉素):inhibit

peptidyl

transferaseineukaryoticribosome,butnotinbacterialribosome

TreatT.aquaticus50SribosomewithErythromycinandAnisomycin

Whatwillhappen?5/7/202645ribosomeErythromycinpeptidyl

transferase

AnisomycinChloramphenicol+++------++-T50ST50S:

Thermus

aquaticus50Sribosomesubunit红霉素茴香霉素氯霉素5/7/202646Questionaboutthepeptidyl

transferasereaction:Whichpartofribosomeisresponsibleforpeptidyl

transferasereaction?(50Ssubunitor30Ssubunit?)rRNAorproteinisrespo