Beilsten-Edmands Victoria, Gordiyenko Yuliya, Kung Jocky Ck, Mohammed Shabaz, Schmidt Carla, Robinson Carol V
Department of Chemistry, University of Oxford , Oxford, UK.
Department of Chemistry, University of Oxford, Oxford, UK; Department of Biochemistry, University of Oxford, Oxford, UK.
Cell Discov. 2015 Aug 11;1:15020. doi: 10.1038/celldisc.2015.20. eCollection 2015.
Translation of messenger RNA (mRNA) into proteins is key to eukaryotic gene expression and begins when initiation factor-2 (eIF2) delivers methionyl initiator tRNA (Met-tRNAi (Met)) to ribosomes. This first step is controlled by eIF2B mediating guanine nucleotide exchange on eIF2. We isolated eIF2 from yeast and used mass spectrometry to study the intact complex, and found that eIF2β is the most labile of the three subunits (eIF2α/β/γ). We then compared conformational dynamics of the ternary complex eIF2:GTP:Met-tRNAi (Met) with apo eIF2 using comparative chemical cross-linking. Results revealed high conformational dynamics for eIF2α in apo eIF2 while in the ternary complex all three subunits are constrained. Novel post-translational modifications identified here in both eIF2 and eIF2B were combined with established sites, and located within protein sequences and homology models. We found clustering at subunit interfaces and highly phosphorylated unstructured regions, at the N-terminus of eIF2β, and also between the eIF2Bε core and catalytic domains. We propose that modifications of these unstructured regions have a key role in regulating interactions between eIF2 and eIF2B, as well as other eIFs.
信使核糖核酸（mRNA）翻译成蛋白质是真核基因表达的关键步骤，起始于起始因子-2（eIF2）将甲硫氨酰起始转运RNA（Met-tRNAi(Met)）递送至核糖体之时。第一步由介导eIF2上鸟嘌呤核苷酸交换的eIF2B控制。我们从酵母中分离出eIF2，并使用质谱法研究完整复合物，发现eIF2β是三个亚基（eIF2α/β/γ）中最不稳定的。然后，我们使用比较化学交联法比较了三元复合物eIF2:GTP:Met-tRNAi(Met)与脱辅基eIF2的构象动力学。结果显示，脱辅基eIF2中的eIF2α具有较高的构象动力学，而在三元复合物中，所有三个亚基均受到限制。我们在此处鉴定出的eIF2和eIF2B中的新型翻译后修饰与已确定的位点相结合，并定位在蛋白质序列和同源模型中。我们发现这些修饰在亚基界面以及高度磷酸化的无结构区域聚集，位于eIF2β的N端，也位于eIF2Bε核心与催化结构域之间。我们提出，这些无结构区域的修饰在调节eIF2与eIF2B以及其他eIF之间的相互作用中起关键作用。
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