Biochemical analysis of the eIF2βγ complex reveals a structural function for eIF2α in catalyzed nucleotide exchange
Biochemical analysis of the eIF2βγ complex reveals a structural function for eIF2α in catalyzed nucleotide exchange
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DOI：
10.1074/jbc.m007398200
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发表时间：
2001-01-12
影响因子：
4.8
通讯作者：
Hannig, EM
中科院分区：
文献类型：
--
作者：
Nika, J;Rippel, S;Hannig, EM
真核翻译起始因子eIF 2是一种异源三聚体，以GTP依赖性方式将Met-tRNA（i）（Met）结合并递送至40 S核糖体亚基。起始需要水解eIF 2结合的GTP，其释放eIF 2 GDP复合物，该复合物通过核苷酸交换因子eIF 2B再循环为GTP形式。eIF 2的Ru亚基通过丝氨酸51的磷酸化在调节核苷酸交换中起关键作用，丝氨酸51将eIF 2转化为eIF 2B催化的交换反应的竞争性抑制剂。我们纯化了一种完全不含α亚基的eIF 2（eIF 2 β γ），以进一步研究eIF 2 α在eIF 2功能中的作用。这些研究利用了一种酵母菌株，该酵母菌株经遗传改变以绕过通常必需的eIF 2 α结构基因（SU 12）的缺失。α-亚基的去除似乎不会显著改变eIF 2在体内与鸟嘌呤核苷酸或Met-tRN（i）（Met）配体的结合。检测43 S起始复合物形成和eIF 5依赖性GTP水解的定性分析显示，eIF 2 β γ和野生型eIF 2异源三聚体之间没有差异。然而，eIF 2B催化的核苷酸交换的稳态动力学分析显示，a亚基的缺失增加了eIF 2 β γ的Km。GDP的一个数量级，V-max的增幅较小。这些数据表明eIF 2 α是eIF 2和eIF 2B之间促进野生型核苷酸交换速率的结构相互作用所必需的。我们认为这种功能有助于α亚基通过可逆磷酸化控制核苷酸交换速率的能力。
Eukaryotic translation initiation factor eIF2 is a heterotrimer that binds and delivers Met-tRNA(i)(Met) to the 40 S ribosomal subunit in a GTP-dependent manner. Initiation requires hydrolysis of eIF2-bound GTP, which releases an eIF2 GDP complex that is recycled to the GTP form by the nucleotide exchange factor eIF2B. The ru-subunit of eIF2 plays a critical role in regulating nucleotide exchange via phosphorylation at serine 51, which converts eIF2 into a competitive inhibitor of the eIF2B-catalyzed exchange reaction. We purified a form of eIF2 (eIF2 beta gamma) completely devoid of the alpha -subunit to further study the role of eIF2 alpha in eIF2 function. These studies utilized a yeast strain genetically altered to bypass a deletion of the normally essential eIF2 alpha structural gene (SU12), Removal of the alpha -subunit did not appear to significantly alter binding of guanine nucleotide or Met-tRN(i)(Met) ligands by eIF2 in vifro. Qualitative assays to detect 43 S initiation complex formation and eIF5-dependent GTP hydrolysis revealed no differences between eIF2 beta gamma and the wild-type eIF2 heterotrimer. However, steady-state kinetic analysis of eIF2B-catalyzed nucleotide exchange revealed that the absence of the a-subunit increased K-m for eIF2 beta gamma (.)GDP by an order of magnitude, with a smaller increase in V-max. These data indicate that eIF2 alpha is required for structural interactions between eIF2 and eIF2B that promote wild-type rates of nucleotide exchange, We suggest that this function contributes to the ability of the alpha -subunit to control the rate of nucleotide exchange through reversible phosphorylation.