REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS

在真核生物中，启动子甲硫酰-tRNA与核糖体的结合是由异三聚体因子eIF2催化的，而在原核生物中，单个多肽因子IF2具有相同的功能。我们已经在古生菌、酵母、酿酒酵母和人类中鉴定并鉴定了IF2同源物。以往的研究表明，酵母IF2同源基因yIF2由FUN12基因编码，是一种通用的翻译启动因子。我们获得了一个人IF2同源物的克隆，我们发现人或古生菌的IF2蛋白可以在体内功能上替代yIF2。此外，人和古生菌的IF2蛋白可以替代yIF2，并刺激Fun12缺失菌株提取液中的蛋白质合成。这些结果表明，IF2是一个普遍保守的翻译因子，蛋白质合成机制在进化过程中比先前预期的更加保守。生化分析表明，人IF2蛋白能促进核糖体亚基参与蛋白质合成。为了认识到这种活性，真核细胞中的IF2同源物被重新命名为eIF5B。使用一个利用XTP代替GTP的eIF5B突变体，我们已经证明在真核翻译起始过程中，亚基的连接至少需要两次核苷酸水解事件。第二个研究兴趣是翻译起始因子eIF2的磷酸化。哺乳动物的PKR和HRI以及酵母酶GCN2特异性地磷酸化eIF2α亚基上的丝氨酸-51，以调节应激条件下的翻译。我们已经证明，痘苗病毒K3L蛋白和猪痘病毒C8L蛋白是PKR的假底物抑制剂，并能抑制PKR在酵母中的毒性。位于eIF2α磷酸化位点约30个残基的序列基序在K3L和C8L中保守，对于抑制酵母和哺乳动物细胞中的PKR是至关重要的。EIF2α这一基序的突变在体内损害了GCN2激酶的磷酸化，表明该基序在底物识别中发挥着重要作用。在PKR激酶结构域的羧基末端一半的14个独立突变使该激酶对K3L抑制具有抵抗力，这些突变被预测会改变该激酶与底物之间的接触。最后，在酵母和哺乳动物细胞中的实验证明了二聚体对活体中PKR激活的重要性。虽然一个分离的PKR激活域在体内是不活跃的，但将该激活域融合到异源二聚化结构域可以恢复活性。-翻译，因子，eIF，激酶，酵母，酿酒酵母

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