IN-VITRO SYNTHESIS OF HUMAN PROTEIN-SYNTHESIS INITIATION FACTOR-4-GAMMA AND ITS LOCALIZATION ON 43-S AND 48-S INITIATION-COMPLEXES

The rate of protein synthesis is controlled in a large number of physi ological situations at the stage of 48 S initiation complex formation, a phase that involves the recruitment of mRNA to the 40 S ribosomal s ubunit. This process is mediated by the eukaryotic initiation factor-4 (eIF-4) group of translation initiation factors consisting of eIF-4E, eIF-4A, eIF-4B, and eIF-4gamma. In order to develop a new tool to stu dy this process, we have produced radiolabeled eIF-4gamma by in vitro transcription and translation. Despite the fact that eIF-4gamma is pre dicted from the cDNA sequence to be 154 kDa, the major synthetic produ ct migrated on SDS-polyacrylamide gel electrophoresis at 205 kDa. Alth ough this is similar to the migration of the fastest polypeptide of au thentic eIF-4gamma (approximately 206 kDa), no products were found to co-migrate with the slowest forms of authentic eIF-4gamma (210-220 kDa ), suggesting that these forms derive from extensive modification of t he initial polypeptide. The in -vitro product also formed a complex wi th eIF-4E, as judged by its ability to bind to m7GTP-Sepharose. Sucros e gradient sedimentation studies demonstrated that eIF-4gamma was pres ent on both 43 and 48 S initiation complexes but not 80 S complexes. T his supports a model in which free eIF-4E binds to mRNA followed by bi nding of the eIF-4E-mRNA complex to a 43 S initiation complex already containing eIF-4gamma.