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dc.contributor.authorChakrabarti, Amitabha
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 53-01, Section: B, page: 9900.
dc.description.abstractStudies on the mechanism of initiation of protein synthesis in eukaryotic cells, particularly with regard to the functions of GTP and protein synthesis initiation factors, eIF-2, eIF-5, eIF-2B, and eIF-3 have been carried out. The function of eIF-5 (M{dollar}\sb{lcub}\rm r{rcub}{dollar} = 58-62 kDa) in the formation of an 80S polypeptide chain initiation complex from a 40S initiation complex has ben investigated. Incubation of the isolated 40S initiation complex (40S{dollar}\cdot{dollar}AUG{dollar}\cdot{dollar}Met{dollar}\cdot{dollar}tRNA{dollar}\sb{lcub}\rm f{rcub}\cdot{dollar}eIF-2 GTP) with eIF-5 resulted in the rapid and quantitative hydrolysis of GTP bound to the 40S initiation complex. Analysis of eIF-5 catalyzed reaction products by gel filtration indicated that both eIF-2{dollar}\cdot{dollar}GDP binary complex and P{dollar}\sb{lcub}\rm i{rcub}{dollar} formed were released from the ribosomal complex while Met-tRNA{dollar}\sb{lcub}\rm f{rcub}{dollar} remained bound to 40S ribosomes as a Met-tRNA{dollar}\sb{lcub}\rm f{rcub}\cdot{dollar}40S{dollar}\cdot{dollar}AUG complex. Reactions carried out with biologically active {dollar}\sp{lcub}32{rcub}{dollar}P-labeled eIF-5 indicated that this protein was not associated with the 40{dollar}\cdot\rm {lcub}AUG{rcub}\cdot\rm {lcub}Met{rcub}{dollar}-tRNA{dollar}\sb{lcub}\rm f{rcub}{dollar} complex; similar results were obtained by immunological methods using monospecific anti-eIF-5 antibodies. The isolated 40S{dollar}\cdot\rm {lcub}AUG{rcub}\cdot{dollar}MetRNA{dollar}\sb{lcub}\rm f{rcub}{dollar} complex, free of eIF-2{dollar}\cdot{dollar}GDP and eIF-5, readily interacted with 60S ribosomal subunits to form the 80S initiation complex capable of transferring Met-tRNA{dollar}\sb{lcub}\rm f{rcub}{dollar} into peptide linkages. In contrast to these results, when 60S ribosomal subunits were present in eIF-5-catalyzed reactions, the eIF-2{dollar}\cdot{dollar}GDP remained bound (albeit weakly) to the 60S ribosomal subunit of the 80S initiation complex. Experiments directed towards understanding the probable role of the 80S initiation complex-bound eIF-2{dollar}\cdot{dollar}GDP have revealed that while purified eIF-2B catalyzes the release of GDP from the eIF-2{dollar}\cdot{dollar}GDP bound to an 80S initiation complex, the complex was unable to cause the release of eIF-2 from the ribosomal complex. In contrast, when 60S ribosomal subunits were preincubated with eIF-2{dollar}\cdot{dollar}eIF-2B complex (the physiological form of eIF-2B), and then added to an 80S initiation reaction mixture, the eIF-2{dollar}\cdot{dollar}GDP produced in the reaction did not bind to the 60S subunit of the 80S initiation complex formed, thus ensuring the release of eIF-2 from the ribosomal complex following the hydrolysis of GTP by eIF-5.;Finally, studies on eIF-5--a factor required for the formation of a 40S initiation complex at physiological 1 mM Mg{dollar}\sp{lcub}2+{rcub}{dollar} concentration have been carried out. Purified eIF-3 consists of 5 major and 3 minor polypeptides of apparent masses of 120, 66, 63, 52, 49, 43, 41 and 26 kDa. Immunochemical characterization of the protein has indicated that eIF-3 comprises of three immunologically distinct polypeptides of 170 kDa, 49 kDa and 43 kDa. The other polypeptides observed in purified eIF-3 preparations are generated by protease degradation of the 170 kDa polypeptide. The specific function of the protein in stimulating the formation of a 40S initiation complex can be reconstituted by allowing only the 49 kDa and 43 kDa polypeptides to renature together.
dc.publisherProQuest Dissertations & Theses
dc.subjectMolecular biology.
dc.titleFunction of protein factors in polypeptide chain initiation in eukaryotes

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