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.

我们研究了真核生物蛋白质合成的机制和调控，重点研究了GTP酶和一系列应激反应蛋白激酶的作用。在蛋白质合成的第一步，翻译起始因子促进80S核糖体在mRNA的AUG密码子上组装。因子eIF2是一种GTP酶，可促进特异性启动子甲硫酰-tRNA(Met-tRNA)与核糖体小亚基的结合。Met-tRNA上的反密码子与mRNA上的Aug密码子的碱基配对触发eIF2对GTP的水解，并从核糖体中释放该因子。因子eIF5B是一种核糖体依赖的GTP酶，促进亚基在翻译起始的最后一步加入。EIF5B是原核生物翻译因子IF2的同源基因。对活性GTP结合和非活性GDP结合的eIF5B的X射线结构的比较表明，伴随GTP水解的因素是杠杆型结构重排。将eIF5B的核苷酸特异性从GTP改变为XTP，导致了有效连接亚基和合成多肽所需的核苷酸。因此，与细菌中单一的GTP翻译启动要求相反，在真核细胞的翻译启动过程中，两个GTP分子被消耗。与生物化学定义的eIF5B在亚基连接中的作用一致，缺乏eIF5B的酵母菌株显示出更高的泄漏扫描水平。我们发现，GTP与eIF5B结合是亚基连接所必需的，而不是eIF5B GTPase活性。EIF5B GTP结合区中普遍保守的Switch 1基序突变会损害细胞生长和eIF5B GTP酶活性，但不会影响亚单位连接。该Switch I突变体的基因内抑制突变恢复到接近野生型生长，但没有恢复该因子的GTP酶活性。这些映射到因子核糖体结合面的抑制突变降低了eIF5B的核糖体结合亲和力。我们认为，GTP结合的eIF5B与40S核糖体预引发复合体结合，在那里它稳定了启动子Met-tRNA与核糖体P位点的结合，并促进了亚基的连接。60S核糖体亚基的加入触发了eIF5B对GTP的水解，并将该因子转化为具有低核糖体结合亲和力的形式。因此，eIF5B是一种调节GTP酶，其中GTP与GDP结合调节因子的核糖体亲和力。 四种蛋白激酶PKR、GCN2、HRI和PERK通过磷酸化eIF2α亚基上的丝氨酸-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的竞争性抑制物。在哺乳动物细胞中，PKR在抗病毒防御中起重要作用，我们在酵母中建立了一个异源系统来研究PKR。PKR的高水平表达抑制了酵母细胞的生长，而PKR的假底物抑制物痘苗病毒K3L蛋白的共表达可以减轻PKR在酵母中的毒性。粘液瘤病毒的M156R蛋白是K3L蛋白的同源物。而K3L蛋白抑制PKR激酶活性，我们发现PKR有效地磷酸化M156R蛋白。在体外，M156R蛋白与eIF2α竞争PKR的磷酸化，提示粘液瘤病毒阻止PKR磷酸化eIF2α的可能机制。在共表达K3L蛋白的酵母中发现了12个单一氨基酸的变化，这些变化恢复了PKR的毒性。我们认为，这些位于PKR激酶域的突变降低了PKR对其假底物的亲和力，而不会严重损害底物结合和磷酸化。N-末端和C-末端截断分析表明，eIF2α的1-180残基是PKR或GCN2有效磷酸化丝氨酸-51的最低底物。在体内和体外都分离到eIF2α残基49、50和79-83的突变，这些突变会损害GCN2和PKR对丝氨酸-51的磷酸化。值得注意的是，丙氨酸取代了天冬氨酸-83，32位残基的磷酸化，完全阻断了磷酸化。我们认为eIF2α激酶利用磷酸化位点附近和远处的残基识别其底物。49、50和79-83位残基上的第二组突变阻止了翻译调控，并可能抑制了eIF2B，但没有抑制丝氨酸-51磷酸化，这表明eIF2α激酶和eIF2B与eIF2α上的重叠表面相互作用。