Function of protein factors in polypeptide chain initiation in eukaryotes

Abstract

Studies 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.