Regulation Of Eukaryotic Protein Synthesis

翻译起始因子eIF5B是原核翻译因子IF2的同源物。IF2的主要功能被认为是将启动物甲硫基trna与核糖体结合，而eIF5B则促进核糖体亚基连接。eIF5B是一种依赖核糖体的GTPase。用x射线晶体学方法对热自养古菌M中eIF5B的三维结构进行了解析。这种蛋白质类似于一个分子圣杯，GTP结合域形成杯子，一个长而坚硬的α -螺旋作为杯柄，一个小的β -桶结构域作为杯底。gtp结合的活性eIF5B和gtp结合的非活性eIF5B的比较表明，gtp结合结构域的微小变化通过涉及长α -螺旋的杠杆型机制被放大，导致c端β -桶结构域的显著移动。酵母eIF5B的gtp结合域的突变分析揭示了eIF5B与gtp结合和水解在翻译起始中的关键作用。利用利用XTP代替GTP的eIF5B突变体，我们证明了真核翻译起始至少需要两个核苷酸（GTP）水解事件。与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。真核翻译因子eIF1A是原核因子IF1的同源物，已知其与核糖体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之间的这种进化上保守的相互作用可能促进启动物甲硫基trna与核糖体P位点的结合。最后，GTP水解和伴随的eIF5B结构域重排可能促进因子释放，Met-tRNA和核糖体调节，使亚基连接。在真核生物中，引发剂甲硫基trna与核糖体的结合是由异源三聚体因子eIF2催化的。哺乳动物激酶PKR、HRI和PERK以及酵母激酶GCN2特异性磷酸化eIF2 α亚基上的丝氨酸-51，以调节应激条件下的翻译。我们证明了牛痘病毒K3L和猪痘病毒C8L蛋白是PKR的假底物抑制剂，并且可以抑制酵母和哺乳动物细胞中的PKR功能。K3L和C8L对PKR的抑制作用依赖于eIF2alpha、K3L和C8L之间保守的残基。目前正在研究抗K3L抑制的PKR突变体。虽然分离的PKR激酶结构域在酵母和哺乳动物细胞中都是失活的，但外源二聚化结构域与PKR激酶结构域的融合恢复了活性。特别值得注意的是，发现GyrB- pkr激酶结构域融合蛋白使eIF2alpha磷酸化，并抑制依赖于药物coumermycin的哺乳动物细胞中的报告基因表达，从而促进GyrB二聚化。酵母eIF2alpha的突变分析显示Ser-51的磷酸化需要1-200个残基。在Ser-51磷酸化位点的49和50残基以及30个残基C端附近的保守序列基序上的氨基酸取代会破坏翻译调控。生化研究表明，在体内和体外，eIF2alpha突变的一个子集阻断了GCN2和PKR激酶的磷酸化。这些结果表明，eIF2alpha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.