Regulation Of Eukaryotic Protein Synthesis

翻译起始因子eIF5B是原核生物翻译因子IF2的同源基因。虽然IF2的主要功能被认为是结合启动子甲硫酰-tRNA与核糖体，而eIF5B促进核糖体亚基的连接。EIF5B是一种依赖核糖体的GTP酶。用X射线结晶学方法研究了自养古生子M的eIF5B的三维结构。该蛋白质类似于一个分子圣杯，其GTP结合结构域形成杯子，一个长而硬的α-螺旋作为茎，一个小的β-桶结构域作为圣杯的底部。对活性的、GTP结合的和非活性的、GDP结合的eIF5B的比较表明，GTP结合结构的微小变化通过涉及长螺旋的杠杆型机制被放大，并导致C末端的β-桶结构的显著移动。对酵母eIF5B的GTP结合域的突变分析表明，eIF5B在GTP结合和eIF5B的翻译起始的水解过程中起着关键作用。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。真核翻译因子eIF1a是原核因子IF1的同源基因，已知能与核糖体A位点结合。我们发现eIF5B和eIF1A在物理和功能上相互作用，促进蛋白质合成和酵母细胞生长。EIF5B/IF2和eIF1A/IF1之间这种进化上保守的相互作用可能有助于启动子甲硫酰-tRNA与核糖体P位点的结合。最后，GTP的水解和eIF5B中伴随的结构域重排可能促进因子释放，以及Met-tRNA和核糖体的调整，从而使亚基能够连接。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。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。我们证明痘苗病毒K3L和猪痘病毒C8L蛋白是PKR的假底物抑制物，在酵母和哺乳动物细胞中都能抑制PKR功能。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。耐K3L抑制的PKR突变体目前正在研究中。然而，在酵母和哺乳动物细胞中，分离的PKR激活域都是无效的，异源二聚化结构域与PKR激活域的融合恢复了活性。特别值得注意的是，在依赖药物香豆素的哺乳动物细胞中，发现了一种GyrB-PKR激活域融合蛋白，可以磷酸化eIF2α并抑制报告基因的表达，而香豆素促进了GyrB的二聚化。酵母eIF2pha的突变分析显示，Ser-51的磷酸化对残基1-200有严格的要求。Ser-51磷酸化位点49和50位氨基酸的替换以及大约30个残基的保守序列基序中的氨基酸替换会损害翻译调控。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。

The translation initiation factor eIF5B is an ortholog of the prokaryotic translation factor IF2. Whereas the primary function of IF2 is thought to be binding the initiator methionyl-tRNA to ribosomes, eIF5B promotes ribosomal subunit joining. The eIF5B is a ribosome-dependent GTPase. The three-dimensional structure of eIF5B from the archaeon M thermoautotrophicum was solved by x-ray crystallography. The protein resembles a molecular chalice with the GTP binding domain forming the cup, a long rigid alpha-helix as the stem, and a small beta-barrel domain as the base of the chalice. Comparison of active, GTP-bound and inactive, GDP-bound eIF5B revealed that minor changes in the structure of the GTP-binding domain were amplified through a lever-type mechanism involving the long alpha-helix and resulted in significant movement of the C-terminal beta-barrel domain. Mutational analysis of the GTP-binding domain of yeast eIF5B 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. The eukaryotic translation factor eIF1A is an ortholog of the prokaryotic factor IF1, that is known to bind to the ribosomal A-site. We have found that eIF5B and eIF1A physically and functionally interact to promote protein synthesis and yeast cell growth. This evolutionarily conserved interaction between eIF5B/IF2 and eIF1A/IF1 may facilitate initiator methionyl-tRNA binding to the ribosomal P site. Finally, GTP hydrolysis and the accompanying domain rearrangements in eIF5B may promote factor release, and Met-tRNA and ribosomal adjustments that enable subunit joining. The binding of initiator methionyl-tRNA to ribosomes is catalyzed in eukaryotic organisms by the heterotrimeric 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. We demonstrated that the vaccinia virus K3L and swine pox virus C8L proteins are pseudosubstrate inhibitors of PKR, and can suppress PKR function in both yeast and mammalian cells. This inhibition of PKR by K3L and C8L was dependent on residues conserved among eIF2alpha, K3L and C8L. PKR mutants resistant to K3L inhibition are currently under investigation. Whereas an isolated PKR kinase domain was inactive in both yeast and mammalian cells, fusion of heterologous dimerization domains to the PKR kinase domain restored activity. Of particular note, a GyrB-PKR kinase domain fusion protein was found to phosphorylate eIF2alpha and inhibit reporter gene expression in mammalian cells dependent on the drug coumermycin, which promotes GyrB dimerization. Mutational analysis of yeast eIF2alpha revealed a stringent requirement for residues 1-200 for phosphorylation of Ser-51. 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 impaired 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.