REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS

在真核生物中，甲硫基trna与核糖体的结合是由异源三聚体因子eIF2催化的，而在原核生物中，单一多肽因子IF2具有相同的功能。我们已经在古细菌、酵母、酿酒酵母和人类中鉴定并鉴定了IF2同源物。先前的研究表明，酵母IF2同源物yIF2是一种通用的翻译起始因子，由FUN12基因编码。我们获得了一个人类IF2同源物的克隆，我们发现人类或古细菌的IF2蛋白在体内可以在功能上替代yIF2。此外，在fun12缺失菌株的提取物中，人类和古细菌的IF2蛋白可以替代yIF2并刺激蛋白合成。这些结果表明，IF2是一个普遍保守的翻译因子，并且蛋白质合成机制在进化过程中比先前预期的更为高度保守。生化实验表明，人IF2蛋白促进了蛋白质合成的核糖体亚基加入步骤。为了认识到这种活性，真核生物IF2同源物被重新命名为eIF5B。利用利用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，亚基连接至少需要两个核苷酸水解事件。第二个研究方向是翻译起始因子eIF2的磷酸化。哺乳动物激酶PKR和HRI以及酵母激酶GCN2特异性磷酸化eIF2 α亚基上的丝氨酸-51，以调节应激条件下的翻译。我们已经证明牛痘病毒K3L蛋白和猪痘病毒C8L蛋白是PKR的假底物抑制剂，可以抑制PKR对酵母的毒性。位于eIF2alpha磷酸化位点约30个残基的序列基序在K3L和C8L中保守，对于酵母和哺乳动物细胞中PKR的抑制至关重要。在体内，eIF2alpha这个基序的突变会破坏GCN2激酶的磷酸化，表明该基序在底物识别中起重要作用。PKR激酶结构域羧基末端的14个独立突变使该激酶抵抗K3L抑制，这些突变预计会改变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化对PKR体内激活的重要性。虽然分离的PKR激酶结构域在体内是无活性的，但发现激酶结构域与异源二聚化结构域的融合可以恢复活性。-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