JournalofVisualizedExperimentswww.jove.comCopyright©2011CreativeCommonsAttribution-NonCommercialLicenseFebruary2011|48|e2498|Page1of6VideoArticleIsolationofTranslatingRibosomesContainingPeptidyl-tRNAsforFunctionalandStructuralAnalysesNitinShirole1,SreeramBalasubramanian1,CharlesYanofsky2,LuisCruz-Vera11DepartmentofBiologicalSciences,UniversityofAlabamaHuntsville2DepartmentofBiology,StanfordUniversityCorrespondenceto:LuisCruz-Veraatlrc0002@uah.eduURL:http://www.jove.com/video/2498DOI:doi:10.3791/2498Keywords:MolecularBiology,Issue48,Ribosomestalling,ribosomeisolation,peptidyl-tRNA,invitrotranslation,RNAchemicalmodification,puromycin,antibiotics.DatePublished:2/25/2011Citation:Shirole,N.,Balasubramanian,S.,Yanofsky,C.,Cruz-Vera,L.IsolationofTranslatingRibosomesContainingPeptidyl-tRNAsforFunctionalandStructuralAnalyses.J.Vis.Exp.(48),e2498,doi:10.3791/2498(2011).AbstractRecently,structuralandbiochemicalstudieshavedetailedmanyofthemoleculareventsthatoccurintheribosomeduringinhibitionofproteinsynthesisbyantibioticsandduringnascentpolypeptidesynthesis.Someoftheseantibiotics,andregulatorynascentpolypeptidesmostlyintheformofpeptidyl-tRNAs,inhibiteitherpeptidebondformationortranslationtermination1-7.Theseinhibitoryeventscanstopthemovementoftheribosome,aphenomenontermed"translationalarrest".Translationarrestinducedbyeitheranantibioticoranascentpolypeptidehasbeenshowntoregulatetheexpressionofgenesinvolvedindiversecellularfunctionssuchascellgrowth,antibioticresistance,proteintranslocationandcellmetabolism8-13.Knowledgeofhowantibioticsandregulatorynascentpolypeptidesalterribosomefunctionisessentialifwearetounderstandthecompleteroleoftheribosomeintranslation,ineveryorganism.Here,wedescribeasimplemethodologythatcanbeusedtopurify,exclusively,foranalysis,thoseribosomestranslatingaspecificmRNAandcontainingaspecificpeptidyl-tRNA14.Thisprocedureisbasedonselectiveisolationoftranslatingribosomesboundtoabiotin-labeledmRNA.Thesetranslationalcomplexesareseparatedfromotherribosomesinthes...
