In vitro synthesis of human protein synthesis initiation factor 4 gamma and its localization on 43 and 48 S initiation complexes.
The rate of protein synthesis is controlled in a large number of physiological situations at the stage of 48 S initiation complex formation, a phase that involves the recruitment of mRNA to the 40 S ribosomal subunit. 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-4 gamma. In order to develop a new tool to study this process, we have produced radiolabeled eIF-4 gamma by in vitro transcription and translation. Despite the fact that eIF-4 gamma is predicted from the cDNA sequence to be 154 kDa, the major synthetic product migrated on SDS-polyacrylamide gel electrophoresis at 205 kDa. Although this is similar to the migration of the fastest polypeptide of authentic eIF-4 gamma (approximately 206 kDa), no products were found to co-migrate with the slowest forms of authentic eIF-4 gamma (210-220 kDa), suggesting that these forms derive from extensive modification of the initial polypeptide. The in vitro product also formed a complex with eIF-4E, as judged by its ability to bind to m7GTP-Sepharose. Sucrose gradient sedimentation studies demonstrated that eIF-4 gamma was present on both 43 and 48 S initiation complexes but not 80 S complexes. This supports a model in which free eIF-4E binds to mRNA followed by binding of the eIF-4E.mRNA complex to a 43 S initiation complex already containing eIF-4 gamma.
Joshi
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B
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport 71130-3932.
Yan
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R
Rhoads
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RE
D002474:Cell-Free System; D004486:Edeine / Q000494:pharmacology; D039561:Eukaryotic Initiation Factor-4E; D006801:Humans; D008715:Methionine / Q000378:metabolism; D016366:Open Reading Frames; D010448:Peptide Initiation Factors / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000187:drug effects; D012314:RNA Cap Analogs / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014158:Transcription, Genetic
D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D012314:RNA Cap Analogs; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; C077382:eIF-4gamma; D004486:Edeine; C017516:7-methylguanosine triphosphate; D008715:Methionine