CP1 domain in Escherichia coli leucyl-tRNA synthetase is crucial for its editing function
CP1 domain in Escherichia coli leucyl-tRNA synthetase is crucial for its editing function
复制标题
大肠杆菌亮氨酰-tRNA 合成酶中的 CP1 结构域对其编辑功能至关重要
DOI：
10.1021/bi000108r
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发表时间：
2000-06-06
期刊：
影响因子：
2.9
通讯作者：
Wang, YL
中科院分区：
文献类型：
--
作者：
Chen, JF;Guo, NN;Wang, YL
氨酰-tRNA合成酶对氨基酸的区分是通过两个筛选步骤实现的;大于同源底物的氨基酸被“粗筛“排除，而小于同源底物的氨基酸的反应产物将通过”细筛”并被水解。在这项研究中，我们创建了LeuRS-B，一个突变的亮氨酰-tRNA合成酶从大肠杆菌与重复的肽片段从Met 328到Pro368（在其CP 1结构域），该突变体具有50%的野生型酶的亮氨酰化活性，并具有相同的能力，区分非同源氨基酸在反应的第一步。然而，LeuRS-B可以催化甲硫氨酸或异亮氨酸对tRNA（Leu）的错误充电，这表明它编辑错误产物的能力受损。野生型亮氨酰-tRNA合成酶可以编辑由LeuRS-B产生的错电荷tRNA（Leu），而分离的CP 1结构域不能。这些数据表明，CP 1结构域的亮氨酰-tRNA合成酶是至关重要的第二个编辑筛，CP 1需要的结构背景亮氨酰-tRNA合成酶，以履行其编辑功能。
The amino acid discrimination by aminoacyl-tRNA synthetase is achieved through two sifting steps; amino acids larger than the cognate substrate are rejected by a "coarse sieve'', while the reaction products of amino acids smaller than the cognate substrate will go through a ''fine sieve" and be hydrolyzed, This "double-sieve" mechanism has been proposed for IIeRS, a class I aminoacyl-tRNA synthetase. In this study, we created LeuRS-B, a mutant leucyl-tRNA synthetase from Escherichia coli with a duplication of the peptide fragment from Met328 to Pro368 (within its CP1 domain), This mutant has 50% of the leucylation activity of the wild-type enzyme and has the same ability to discriminate noncognate amino acids in the first step of the reaction. However, LeuRS-B can catalyze mischarging of tRNA(Leu) by methionine or isoleucine, suggesting that it is impaired in the ability to edit incorrect products. Wild-type leucyl-tRNA synthetase can edit the mischarged tRNA(Leu) made by LeuRS-B, while a separated CP1 domain cannot. These data suggest that the CP1 domain of leucyl-tRNA synthetase is crucial to the second editing sieve and that CP1 needs the structural context in leucyl-tRNA synthetase to fulfill its editing function.