Please use this identifier to cite or link to this item: https://hdl.handle.net/20.500.12202/3227
Title: Initiation factors in eukaryotic protein synthesis
Authors: Ghosh, Sankar
Keywords: Molecular biology.
Issue Date: 1988
Publisher: ProQuest Dissertations & Theses
Citation: Source: Dissertation Abstracts International, Volume: 49-08, Section: B, page: 3020.;Advisors: Umadas Maitra.
Abstract: A protein factor, designated Guanine nucleotide Exchange Factor (GEF), has been isolated and purified from post ribosomal supernatants of rabbit reticulocyte lysates and calf liver extracts, that can displace GDP from an eIF2.GDP complex in the presence of GTP. The purification of the factor was based on a direct assay of its function, i.e. the formation of an eIF2.GTP.Met tRNA{dollar}\sb{lcub}\rm f{rcub}{dollar} ternary complex from an eIF2.GDP complex. Detailed studies of the properties of eIF2.GDP complex and GEF catalyzed exchange of GDP by GTP have shown that while both the hetrodimer ({dollar}\alpha\gamma{dollar}) eIF2 as well as the heterotrimer ({dollar}\alpha\beta\gamma{dollar}) eIF2 are active in 80S initiation complex formation, GEF catalyzed displacement of GDP from an eIF2.GDP complex in the presence of GTP, occurs only with the heterotrimeric ({dollar}\alpha\beta\gamma{dollar}) form of eIF2. These results indicate that the {dollar}\beta{dollar} subunit of eIF2 is essential for catalytic reutilization of eIF2 in initiation reactions. We have also demonstrated that the inhibition of eIF2.GDP binary complex formation and eIF2.GTP.Met tRNA{dollar}\sb{lcub}\rm f{rcub}{dollar} ternary complex formation observed by inclusion of 1 mM Mg{dollar}\sp{lcub}2+{rcub}{dollar} in reaction mixtures is due to the presence of endogenous GDP in purified eIF2 preparations. We have devised procedures that lead to the isolation of purified eIF2 preparations that are free of endogenously bound GDP. The activity of such eIF2 preparations, free of endogenously bound GDP, are no longer inhibited by inclusion of Mg{dollar}\sp{lcub}2+{rcub}{dollar} in reaction mixtures.;In addition to the above studies, the protein factor eIF5 has been further characterized. Polyclonal antibodies were raised against denatured eIF5 in rabbits. When Hela or mouse L cells were lysed rapidly into denaturing buffer containing SDS and the lysates were analyzed by polyacrylamide gel electrophoresis, followed by immunoblotting with anti eIF5 antibodies, an intense band developed which corresponded in molecular weight with purified eIF5. The results indicate that isolated eIF5 is of the same size as that found in crude lysates. In addition, the antibodies were also used to screen a recombinant {dollar}\lambda{dollar} gt11 rat brain cDNA library and several positive clones identified and sequenced.
URI: https://ezproxy.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:8822209
https://hdl.handle.net/20.500.12202/3227
Appears in Collections:Albert Einstein College of Medicine: Doctoral Dissertations

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