REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS

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The binding of initiator methionyl-tRNA to ribosomes is catalyzed in eukaryotic organisms by the heterotrimeric factor eIF2, whereas in prokaryotes a single polypeptide factor IF2 performs the same function. We have identified and characterized IF2 homologs in archaea, the yeast Saccharomyces cerevisiae and humans. Previous studies demonstrated that the yeast IF2 homolog yIF2, encoded by the FUN12 gene, is a general translation initiation factor. We obtained a clone for a human IF2 homolog, and we found that human or archaeal IF2 proteins could functionally substitute in vivo for yIF2. In addition, the human and archaeal IF2 proteins could substitute for yIF2 and stimulate protein synthesis in extracts from fun12-deletion strains. These results demonstrate that IF2 is a universally conserved translation factor and that the mechanism of protein synthesis has been more highly conserved during evolution than previously anticipated. Biochemical assays demonstrated that the human IF2 protein promotes the ribosomal subunit joining step of protein synthesis. In recognition of this activity the eukaryotic IF2 homologs have been renamed eIF5B. Using an eIF5B mutant that utilizes XTP in place of GTP, we have demonstrated that at least two nucleotide hydrolysis events are required for subunit joining during eukaryotic translation initiation. A second research interest is phosphorylation of the translation initiation factor eIF2. The mammalian kinases PKR and HRI and the yeast kinase GCN2 specifically phosphorylate serine-51 on the alpha subunit of eIF2 to regulate translation during stress conditions. We have demonstrated that the vaccinia virus K3L protein and the swine pox virus C8L protein are pseudosubstrate inhibitors of PKR, and can suppress PKR toxicity in yeast. A sequence motif located around 30 residues from the site of phosphorylation in eIF2alpha and conserved in K3L and C8L was critical for the inhibition of PKR in both yeast and mammalian cells. Mutations in this motif of eIF2alpha impaired phosphorylation by the GCN2 kinase in vivo, indicating that motif plays an important role in substrate recognition. Fourteen independent mutations in the carboxyl- terminal half of the PKR kinase domain rendered the kinase resistant to K3L inhibition, and these mutations are predicted to alter contacts between the kinase and substrate. Finally, experiments in yeast and mammalian cells demonstrated the importance of dimerization for PKR activation in vivo. Whereas an isolated PKR kinase domain was inactive in vivo, fusion of the kinase domain to heterologous dimerization domains was found to restore activity. - translation, factor, eIF, kinase, yeast, Saccharomyces cerevisiae