Gomez Edith, Mohammad Sarah S, Pavitt Graham D
Biomolecular Sciences, University of Manchester Institute of Science and Technology, Manchester M60 1QD, UK.
EMBO J. 2002 Oct 1;21(19):5292-301. doi: 10.1093/emboj/cdf515.
For protein synthesis initiation in eukaryotes, eIF2B is the guanine-nucleotide exchange factor for eIF2. eIF2B is an essential multi-subunit factor and a major target for translational control in both yeast and mammalian cells. It was shown previously that the largest eIF2B subunit, eIF2Bepsilon, is the only single subunit with catalytic function. Here we report the results of a molecular dissection of the yeast epsilon subunit encoded by GCD6 in which we have identified the catalytic domain. By analysis of a series of N-terminal deletions in vitro we find that the smallest catalytically active fragment contains residues 518-712 (termed Gcd6p(518-712)). Further deletion to position 581 (Gcd6p(581-712)) results in loss of nucleotide exchange function, but eIF2-binding activity is retained. C- terminal deletion of only 61 residues (Gcd6p(1-651)) results in loss of both functions. Thus Gcd6p(518-712) contains two regions that together constitute the catalytic domain of eIF2B. Finally, we show that the catalytic domain can provide eIF2B biological function in vivo when elevated levels eIF2 and tRNA(i)(Met) are also present.
在真核生物中，对于蛋白质合成起始而言，真核起始因子2B（eIF2B）是真核起始因子2（eIF2）的鸟嘌呤核苷酸交换因子。eIF2B是一种必需的多亚基因子，也是酵母和哺乳动物细胞中翻译控制的主要靶点。先前的研究表明，eIF2B最大的亚基eIF2Bε是唯一具有催化功能的单个亚基。在此，我们报告了对由GCD6编码的酵母ε亚基进行分子剖析的结果，其中我们鉴定出了催化结构域。通过对一系列N端缺失进行体外分析，我们发现最小的具有催化活性的片段包含518 - 712位的氨基酸残基（称为Gcd6p(518 - 712)）。进一步缺失至581位（Gcd6p(581 - 712)）会导致核苷酸交换功能丧失，但仍保留eIF2结合活性。仅C端缺失61个残基（Gcd6p(1 - 651)）会导致两种功能均丧失。因此，Gcd6p(518 - 712)包含两个区域，它们共同构成了eIF2B的催化结构域。最后，我们表明当也存在高水平的eIF2和起始甲硫氨酰 - tRNA（tRNA(i)(Met)）时，催化结构域能够在体内提供eIF2B的生物学功能。
