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同源基因由FUN12基因编码，是一种通用的翻译启动因子。生化分析表明，人IF2蛋白具有核糖体依赖的GTP酶活性，并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。EIF5B GTP结合区保守残基的突变揭示了eIF5B对GTP结合和eIF5B的翻译起始作用的关键作用。使用一个利用XTP代替GTP的eIF5B突变体，我们已经证明了真核翻译启动至少需要两个核苷酸(GTP)水解事件。缺乏eIF5B的酵母菌株表现出更高的泄漏扫描水平，这与亚基连接中的作用一致。酵母双杂交、体外蛋白结合实验和免疫共沉淀实验表明，酵母eIF5B与翻译因子eIF1a(原核因子IF1的同源物)直接相互作用。此外，eIF1a的过表达特异性地加剧了缺乏或表达截短形式eIF5B的菌株的生长缺陷。这两个进化上保守的翻译起始因子之间的这种物理和功能上的相互作用可能促进甲硫酰-tRNA与核糖体P位点的结合。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、HRI、PERK和酵母酶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。对酵母eIF2pha的突变分析发现，在Ser-51磷酸化位点的49和50位残基以及大约30个残基的保守序列基序中存在氨基酸替换，这削弱了翻译调控。生化研究表明，在体内和体外，eIF2α突变的一部分阻断了GCN2和PKR激酶的磷酸化。这些结果表明，eIF2α的激酶识别利用了磷酸化位点附近的残基，令人惊讶的是，也利用了远离磷酸化位点的残基。我们证明了痘苗病毒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。K3L和C8L对PKR的这种抑制作用依赖于eIF2α、K3L和C8L之间保守的残基。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。

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, encoded by the FUN12 gene, is a general translation initiation factor. Biochemical assays demonstrated that the human IF2 protein possesses ribosome-dependent GTPase activity and promotes the ribosomal subunit joining step of protein synthesis. In recognition of this activity the eukaryotic IF2 homologs have been renamed eIF5B. Mutation of conserved residues in the eIF5B GTP-binding domain revealed a critical role for GTP-binding and hydrolysis by eIF5B for translation initiation. Using an eIF5B mutant that utilizes XTP in place of GTP, we have demonstrated that at least two nucleotide (GTP) hydrolysis events are required for eukaryotic translation initiation. Consistent with a role in subunit joining, yeast strains lacking eIF5B show increased levels of leaking scanning. Finally, yeast two-hybrid, in vitro protein binding assays and co-immunoprecipitation experiments revealed that yeast eIF5B directly interacts with the translation factor eIF1A (a homolog of the prokaryotic factor IF1). In addition, overexpression of eIF1A specifically exacerbated the growth defect of strains lacking, or expressing truncated forms of, eIF5B. This physical and functional interaction between the two evolutionarily conserved translation initiation factors may facilitate methionyl-tRNA binding to the ribosomal P site.A second research interest is phosphorylation of the translation initiation factor eIF2. The mammalian kinases PKR, HRI, and PERK and the yeast kinase GCN2 specifically phosphorylate serine-51 on the alpha subunit of eIF2 to regulate translation during stress conditions. Mutational analysis of yeast eIF2alpha identified amino acid substitutions at residues 49 and 50 as well as in a conserved sequence motif around 30 residues C-terminal of the Ser-51 phosphorylation site that impair translational regulation. Biochemical studies revealed that a subset of the mutations in eIF2alpha blocked phosphorylation by the GCN2 and PKR kinases both in vivo and in vitro. These results demonstrate that kinase recognition of eIF2alpha utilizes residues both nearby and, surprisingly, remote from the phosphorylation site. We 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. This inhibition of PKR by K3L and C8L was dependent on residues conserved among eIF2alpha, K3L and C8L. 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.