Binding and release of radiolabeled eukaryotic initiation factors 2 and 3 during 80 S initiation complex formation.
The AUG-dependent formation of an 80 S ribosomal initiation complex was studied using purified rabbit reticulocyte initiation factors radiolabeled by reductive methylation. The radiolabeled initiation factors were as biologically active as untreated factors. Reaction mixtures containing a variety of components (AUG, GTP, Met-tRNAf, initiation factors, and 40 S and 60 S ribosomal subunits) were incubated at 30 degrees C and then analyzed on linear sucrose gradients for the formation of ribosomal complexes. The results show that both eukaryotic initiation factor (eIF)-3 and the ternary complex (eIF-2.GTP.Met-tRNAf) bind independently to the 40 S subunit and each of these components enhances the binding of the other. All of the polypeptides of eIF-2 and eIF-3 participate in this binding. Formation of an 80 S ribosomal complex requires eIF-5 and 60 S subunits in a reaction that is stimulated by eIF-4C. Both eIF-2 and eIF-3 are released from the 40 S preinitiation complex during formation of the 80 S initiation complex. Release of eIF-2 and eIF-3 does not occur and 80 S ribosomal complexes are not formed if GTP is replaced by a nonhydrolyzable analog such as guanosine 5'-O3-(1,2-mu-imido)triphosphate. Despite a variety of attempts, it has not yet been possible to demonstrate binding of eIF-4C, eIF-4D, or eIF-5 to either 40 S or 80 S ribosomal complexes.
D000818:Animals; D007700:Kinetics; D008715:Methionine; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism*; D011485:Protein Binding; D012343:RNA, Transfer / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
D010448:Peptide Initiation Factors; D012343:RNA, Transfer; D008715:Methionine