Kapp Lee D, Lorsch Jon R
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street 625 WBSB, Baltimore, MD 21205-2185, USA.
J Mol Biol. 2004 Jan 23;335(4):923-36. doi: 10.1016/j.jmb.2003.11.025.
Eukaryotic translation initiation factor 2 (eIF2) is a G-protein that functions as a central switch in the initiation of protein synthesis. In its GTP-bound state it delivers the methionyl initiator tRNA (Met-tRNA(i)) to the small ribosomal subunit and releases it upon GTP hydrolysis following the recognition of the initiation codon. We have developed a complete thermodynamic framework for the assembly of the Saccharomyces cerevisiae eIF2.GTP.Met-tRNA(i) ternary complex and have determined the effect of the conversion of GTP to GDP on eIF2's affinity for Met-tRNA(i) in solution. In its GTP-bound state the factor forms a positive interaction with the methionine moiety on Met-tRNA(i) that is disrupted when GTP is replaced with GDP, while contacts between the factor and the body of the tRNA remain intact. This positive interaction with the methionine residue on the tRNA may serve to ensure that only charged initiator tRNA enters the initiation pathway. The toggling on and off of the factor's interaction with the methionine residue is likely to play an important role in the mechanism of initiator tRNA release upon initiation codon recognition. In addition, we show that the conserved base-pair A1:U72, which is known to be a critical identity element distinguishing initiator from elongator methionyl tRNA, is required for recognition of the methionine moiety by eIF2. Our data suggest that a role of this base-pair is to orient the methionine moiety on the initiator tRNA in its recognition pocket on eIF2.
真核生物翻译起始因子2（eIF2）是一种G蛋白，在蛋白质合成起始过程中起核心开关的作用。在其结合GTP的状态下，它将甲硫氨酰起始tRNA（Met-tRNA(i)）递送至小核糖体亚基，并在起始密码子被识别后随着GTP水解而释放它。我们已经建立了一个完整的热力学框架来研究酿酒酵母eIF2.GTP.Met-tRNA(i)三元复合物的组装，并确定了GTP向GDP的转化对eIF2在溶液中与Met-tRNA(i)亲和力的影响。在其结合GTP的状态下，该因子与Met-tRNA(i)上的甲硫氨酸部分形成正向相互作用，当GTP被GDP取代时这种相互作用被破坏，而该因子与tRNA主体之间的接触保持完整。与tRNA上甲硫氨酸残基的这种正向相互作用可能有助于确保只有携带氨基酸的起始tRNA进入起始途径。该因子与甲硫氨酸残基相互作用的开启和关闭可能在起始密码子识别后起始tRNA释放的机制中起重要作用。此外，我们表明保守碱基对A1:U72（已知是区分起始甲硫氨酰tRNA和延伸甲硫氨酰tRNA的关键识别元件）是eIF2识别甲硫氨酸部分所必需的。我们的数据表明，这个碱基对的作用是将起始tRNA上的甲硫氨酸部分定位在eIF2上其识别口袋中。
