Mechanism and Regulation Of Eukaryotic Protein Synthesis

我们研究真核生物蛋白质合成的机制和调控，重点是GTP酶和应激反应蛋白激酶家族的作用。在蛋白质合成的第一步中，翻译起始因子促进80 S核糖体在mRNA的AUG密码子处的组装。因子eIF 2是促进特异性引发剂甲硫氨酰-tRNA（Met-tRNA）与小核糖体亚基结合的GT3。Met-tRNA上的反密码子与mRNA上的AUG密码子的碱基配对触发eIF 2水解GTP，并从核糖体释放该因子。因子eIF 5 B是一种核糖体依赖性GT3，其在翻译起始的最后步骤中促进亚基连接。eIF 5 B是原核翻译因子IF 2的直系同源物。活性GTP结合和非活性GDP结合的eIF 5 B的X射线结构的比较揭示了伴随GTP水解的因子中的α-型结构域重排。将eIF 5 B的核苷酸特异性从GTP改变为XTP导致需要两种核苷酸用于有效的亚基连接和肽合成。因此，与细菌中翻译起始需要单个GTP相反，真核翻译起始期间消耗两个GTP分子。与eIF 5 B在亚基连接中的生物化学定义的作用一致，缺乏eIF 5 B的酵母菌株显示出增加的泄漏扫描水平。我们发现，GTP结合eIF 5 B，但不是eIF 5 B GTdR活性，是必不可少的亚基连接。eIF 5 B GTP结合结构域中普遍保守的开关1基序突变会损害细胞生长和eIF 5 B GTP酶活性，但不会损害亚基连接。该开关I突变体的基因内抑制突变恢复了接近野生型的生长，但没有恢复该因子的GTdR活性。这些抑制突变，映射到核糖体结合的因素，降低了核糖体结合亲和力的eIF 5 B。我们建议GTP结合的eIF 5 B结合到40 S核糖体preinitiation复合物，在那里它稳定的启动器Met-tRNA的核糖体P位点的结合，并促进亚基连接。60 S核糖体亚基的连接触发eIF 5 B水解GTP，并将该因子转化为具有低核糖体结合亲和力的形式。因此，eIF 5 B是一种调节性GTP酶，其中GTP与GDP的结合决定了该因子的核糖体亲和力。 四种蛋白激酶PKR、GCN 2、HRI和PERK通过磷酸化eIF 2 α亚基上的丝氨酸-51来调节翻译，将eIF 2从底物转化为其鸟嘌呤核苷酸交换因子eIF 2B的竞争性抑制剂。激酶PKR在哺乳动物细胞中参与抗病毒防御，我们在酵母中建立了一个异源系统来研究PKR。PKR的高水平表达抑制酵母细胞生长，并且痘苗病毒K3 L蛋白（PKR的假底物抑制剂）的共表达增强了PKR在酵母中的毒性。来自粘液瘤病毒的M156 R蛋白是K3 L蛋白的同源物。尽管K3 L蛋白抑制PKR激酶活性，但我们发现PKR有效地磷酸化M156 R蛋白。M156 R蛋白在体外与eIF 2 α竞争PKR磷酸化，提示粘液瘤病毒阻止PKR磷酸化eIF 2 α的可能机制。在PKR激酶结构域中鉴定了12个单个氨基酸的变化，其恢复了共表达K3 L蛋白的酵母中的PKR毒性。我们认为，这些突变，位于PKR激酶结构域，降低PKR的亲和力，其假底物，而不严重损害底物结合和磷酸化。N-和C-末端截短分析显示，eIF 2 α的残基1-180代表PKR或GCN 2有效磷酸化丝氨酸-51的最小底物。在eIF 2 α残基49、50和79-83中分离出突变，这些突变在体内和体外均损害GCN 2和PKR对丝氨酸-51的磷酸化。引人注目的是，丙氨酸取代天冬氨酸-83，32个残基的磷酸化位点，完全阻断磷酸化。我们建议eIF 2 α激酶识别其底物利用附近和远离磷酸化位点的残基。在残基49、50和79-83中鉴定出第二组突变，其阻断翻译调节，并且可能是eIF 2B抑制，但不是丝氨酸-51磷酸化，这表明eIF 2 α激酶和eIF 2B与eIF 2 α上的重叠表面相互作用。

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.