Mechanism and Regulation Of Eukaryotic Protein Synthesis

真核蛋白质合成机制及调控

• 批准号: 6813692
• 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/6813692
• 关键词: Saccharomyces cerevisiae; X ray crystallography; enzyme activity; eukaryote; genetic regulation; genetic translation; guanosine triphosphate; guanosinetriphosphatases; phosphorylation; protein biosynthesis; protein kinase; protein structure function; protein tyrosine kinase; ribosomes; suppressor mutations; transfer RNA; translation factor

We study the mechanism and regulation of eukaryotic protein synthesis focusing on the roles of GTPases and a family of stress-responsive protein kinases. In the first step of protein synthesis translation initiation factors promote the assembly of an 80S ribosome at the AUG codon of an mRNA. The factor eIF2 is a GTPase that facilitates binding of the specific initiator methionyl-tRNA (Met-tRNA) to the small ribosomal subunit. Base-pairing of the anticodon on the Met-tRNA with an AUG codon on an mRNA triggers GTP hydrolysis by eIF2, and release of the factor from the ribosome. The factor eIF5B is a ribosome-dependent GTPase that promotes subunit joining in the final step of translation initiation. The eIF5B is an ortholog of the prokaryotic translation factor IF2. Comparison of the X-ray structures of active GTP-bound and inactive GDP-bound eIF5B revealed lever-type domain rearrangements in the factor accompanying GTP hydrolysis. Changing the nucleotide specificity of eIF5B from GTP to XTP resulted in the requirement for both nucleotides for efficient subunit joining and peptide synthesis. Thus, in contrast to the single GTP requirement for translation initiation in bacteria, two GTP molecules are consumed during eukaryotic translation initiation. Consistent with the biochemically-defined role for eIF5B in subunit joining, yeast strains lacking eIF5B showed increased levels of leaking scanning. We found that GTP-binding to eIF5B, but not eIF5B GTPase activity, was essential for subunit joining. Mutation of the universally conserved Switch 1 motif in the eIF5B GTP-binding domain impaired cell growth and eIF5B GTPase activity, but not subunit joining. Intragenic suppressor mutations of this Switch I mutant restored near wild-type growth, but did not restore the GTPase activity of the factor. These suppressor mutations, which map to the ribosome-binding face of the factor, lowered the ribosome-binding affinity of eIF5B. We propose that GTP-bound eIF5B binds to 40S ribosome preinitiation complexes, where it stabilizes binding of the initiator Met-tRNA to the ribosomal P site and promotes subunit joining. Joining of the 60S ribosomal subunit triggers GTP hydrolysis by eIF5B, and coverts the factor into a form with low ribosome binding affinity. Thus, eIF5B is a regulatory GTPase in which GTP versus GDP binding governs the ribosomal affinity of the factor. Four protein kinases PKR, GCN2, HRI and PERK regulate translation by phosphorylating serine-51 on the alpha subunit of eIF2, converting eIF2 from a substrate to a competitive inhibitor of its guanine nucleotide exchange factor eIF2B. The kinase PKR contributes to anti-viral defense in mammalian cells, and we established a heterologous system in yeast to study PKR. High-level expression of PKR inhibits yeast cell growth, and co-expression of the vaccinia virus K3L protein, a pseudosubstrate inhibitor of PKR, alleviates PKR toxicity in yeast. The M156R protein from myxoma virus is a homolog of the K3L protein. Whereas the K3L protein inhibits PKR kinase activity, we found that PKR efficiently phosphorylates the M156R protein. The M156R protein competed with eIF2alpha for phosphorylation by PKR in vitro, suggesting a possible mechanism by which myxoma virus prevents PKR phosphorylation of eIF2alpha. Twelve single amino acid changes were identified in the PKR kinase domain that restored PKR toxicity in yeast co-expressing the K3L protein. We propose that these mutations, located in the PKR kinase domain, lower the affinity of PKR for its pseudosubstrate without severely impairing substrate binding and phosphorylation. N- and C-terminal truncation analyses revealed that residues 1-180 of eIF2alpha represent the minimal substrate for efficient phosphorylation of serine-51 by PKR or GCN2. Mutations were isolated in eIF2alpha residues 49, 50, and 79-83 that impaired phosphorylation of serine-51 by GCN2 and PKR both in vivo and in vitro. Strikingly, substitution of alanine for aspartic acid-83, 32 residues from the site of phosphorylation, completely blocked phosphorylation. We propose that the eIF2alpha kinases recognize their substrate utilizing residues both nearby and remote from the phosphorylation site. The identification of a second set of mutations in residues 49, 50 and 79-83 that block translational regulation, and presumably eIF2B inhibition, but not serine-51 phosphorylation indicates that the eIF2alpha kinases and eIF2B interact with overlapping surfaces on eIF2alpha.

我们研究了真核蛋白合成的机制和调节，重点是GTPase和一系列应激反应性蛋白激酶的作用。在蛋白质合成翻译起始因子的第一步中，在mRNA的Aug密码子上促进了80S核糖体的组装。 EIF2因子是一种GTPase，可促进特定起始蛋白基-TRNA（Met-tRNA）与小核糖体亚基的结合。用eIF2触发mRNA的GTP水解，在mET-tRNA上对反密码子的碱基对底型，并从核糖体中释放因子。 EIF5B因子是核糖体依赖性GTPase，在翻译起始的最后一步中促进了亚基。 EIF5b是原核翻译因子IF2的直系同源物。比较活跃的GTP结合和不活跃的GDP结构EIF5B的X射线结构，显示伴随GTP水解的因子中的杠杆型结构域重排。将EIF5B的核苷酸特异性从GTP更改为XTP，导致对两个核苷酸的有效亚基连接和肽合成的要求。因此，与细菌翻译起始的单个GTP要求相反，在真核翻译开始期间消耗了两个GTP分子。与EIF5B在亚基连接中的生化作用一致，缺乏EIF5B的酵母菌菌株显示泄漏扫描水平增加。我们发现，GTP结合到EIF5B而不是EIF5B GTPase活性，对于亚基加入至关重要。 EIF5B GTP结合结构域中普遍保守的开关1基序的突变受损细胞的生长和EIF5B GTPase活性，但没有亚基连接。该开关I突变体的基因内抑制剂突变恢复了野生型生长，但没有恢复该因子的GTPase活性。这些抑制剂突变映射到因子的核糖体结合面，降低了EIF5B的核糖体结合亲和力。我们提出，与GTP结合的EIF5B结合了40S核糖体预启发络合物，在该复合物中，它稳定了引发剂Met-tRNA与核糖体P位点的结合并促进亚基连接。 60s核糖体亚基的连接触发了EIF5B的GTP水解，并将该因子掩盖为具有低核糖体结合亲和力的形式。因此，EIF5B是一种调节性GTPase，其中GTP与GDP结合控制了该因子的核糖体亲密关系。 四种蛋白激酶PKR，GCN2，HRI和PERK通过在EIF2的α亚基上磷酸化丝氨酸51来调节翻译，将EIF2从底物转化为其鸟嘌呤核苷酸交换因子EIF2B的竞争抑制剂。激酶PKR有助于哺乳动物细胞的抗病毒防御，我们在酵母中建立了一个异源系统以研究PKR。 PKR的高水平表达抑制酵母细胞的生长，并共表达PKR的假基底抑制剂，牛ac病毒K3L蛋白会减轻酵母中的PKR毒性。来自粘液瘤病毒的M156R蛋白是K3L蛋白的同源物。 K3L蛋白抑制PKR激酶活性，但我们发现PKR有效地磷酸化了M156R蛋白。 M156R蛋白与EIF2Alpha竞争PKR在体外磷酸化，这表明粘液瘤病毒可防止EIF2Alpha的PKR磷酸化。在PKR激酶结构域中鉴定了十二个单氨基酸变化，该域在酵母中恢复了共表达K3L蛋白的PKR毒性。我们提出，这些突变位于PKR激酶结构域中，降低了PKR对其伪造物的亲和力，而不会严重损害底物结合和磷酸化。 N-和C末端截断分析表明，eIF2Alpha的残基1-180代表PKR或GCN2对丝氨酸51有效磷酸化的最小底物。在EIF2Alpha残基49、50和79-83中分离突变，这些突变在体内和体外都受到GCN2和PKR的丝氨酸51损害。令人惊讶的是，丙氨酸代替天冬氨酸83，磷酸化部位的32个残基完全阻​​断了磷酸化。我们建议eIF2Alpha激酶识别其底物利用附近和距磷酸化位点的遥控物的残基。鉴定残基49、50和79-83中的第二组突变，这些突变阻断了转化调节，并且可能是EIF2B抑制，但丝氨酸-51磷酸化可能表明EIF2Alpha激酶和EIF2B在EIF2Alpha上与重叠的表面相互作用。