REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS

真核蛋白质合成的调控

• 批准号: 6290205
• 负责人: THOMAS E DEVER
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6290205
• 关键词: Baculoviridae; eukaryote; gene induction /repression; gene mutation; genetic regulation; genetic strain; oxidative stress; phosphorylation; protein biosynthesis; protein kinase; protein tyrosine kinase; translation factor; vaccinia virus; yeasts

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

起始蛋白基TRNA与核糖体的结合是通过异三聚体因子EIF2催化在真核生物中的，而在原核生物中，单个多肽因子在单个多肽因子中发挥了相同的功能。我们已经确定并表征了古细菌，酵母糖酵母和人类的IF2同源物。先前的研究表明，由FUN12基因编码的酵母IF2同源性YIF2是一般翻译起始因子。我们获得了人类IF2同源物的克隆，我们发现人类或古细菌IF2蛋白可以在体内替代YIF2。另外，人类和古细菌IF2蛋白可以代替YIF2并刺激Fun12-损坏菌株提取物中的蛋白质合成。这些结果表明，IF2是一种普遍保守的翻译因子，并且在进化过程中蛋白质合成的机理比以前预期的要高。生化测定表明，人IF2蛋白会促进核糖体亚基连接蛋白质合成的步骤。为了认识到这项活动，真核IF2同源物已更名为EIF5B。使用利用XTP代替GTP的EIF5B突变体，我们证明了在真核翻译起始中至少需要两个核苷酸水解事件。第二个研究兴趣是翻译起始因子EIF2的磷酸化。哺乳动物激酶PKR和HRI以及酵母激酶GCN2在EIF2的α亚基上特异性磷酸化丝氨酸-51磷酸化，以调节应力条件下的翻译。我们已经证明，谷谷病毒K3L蛋白和猪痘病毒C8L蛋白是PKR的伪基底抑制剂，并且可以抑制酵母中的PKR毒性。来自EIF2Alpha磷酸化位点的约30个残基的序列基序，在K3L和C8L中保守的序列对于抑制酵母和哺乳动物细胞的PKR至关重要。 EIF2Alpha基序中的突变损害了体内GCN2激酶的磷酸化，表明基序在底物识别中起重要作用。 PKR激酶结构域的羧基末端中的14个独立突变使激酶对K3L抑制作用产生了抗性，并且预计这些突变会改变激酶与底物之间的接触。最后，酵母和哺乳动物细胞的实验证明了二聚化对体内PKR激活的重要性。尽管分离的PKR激酶结构域在体内不活跃，但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