Function of eukaryotic initiation factor 5 in the formation of an 80 S ribosomal polypeptide chain initiation complex

Eukaryotic initiation factor 5 (eIF-5), isolated from rabbit reticulocyte lysates, is a monomeric protein of 58-62 kDa. The function of eIF-5 in the formation of an 80 S polypeptide chain initiation complex from a 40 S initiation complex has been investigated. Incubation of the isolated 40 S initiation complex (40 S·AUG·Met·tRNA(f)·eIF-2 GTP) with eIF-5 resulted in the rapid and quantitative hydrolysis of GTP bound to the 40 S initiation complex. The rate of this reaction was unaffected by the presence of 60 S ribosomal subunits. Analysis of eIF-5-catalyzed reaction products by gel filtration indicated that both eIF-2·GDP binary complex and P(i) formed were released from the ribosomal complex whereas Met-tRNA(f) remained bound to 40 S ribosomes as a Met-tRNA(f)·40 S·AUG complex. Reactions carried out with biologically active ³²P-labeled eIF-5 indicated that this protein was not associated with the 40 S·AUG·Met-tRNA(f) complex; similar results were obtained by immunological methods using monospecific anti-eIF-5 antibodies. The isolated 40 S·AUG·Met-RNA(f) complex, free of eIF-2·GDP binary complex and eIF-5, readily interacted with 60 S ribosomal subunits in the absence of exogenously added eIF-5 to form the 80 S initiation complex capable of transferring Met-tRNA(f) into peptide linkages. These results indicate that the sole function of eIF-5 in the initiation of protein synthesis is to mediate hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. This leads to formation of the intermediate 40 S·AUG·Met-tRNA(f) and dissociation of the eIF-2·GDP binary complex. Subsequent joining of 60 S ribosomal subunits to the intermediate 40 S·AUG·Met-tRNA(f) complex does not require participation of eIF-5. Thus, the formation of an 80 S ribosomal polypeptide chain initiation complex from a 40 S ribosomal initiation complex can be summarized by the following sequence of partial reactions.