A conserved threonine within Escherichia coli leucyl-tRNA synthetase prevents hydrolytic editing of leucyl-tRNALeu
A conserved threonine within Escherichia coli leucyl-tRNA synthetase prevents hydrolytic editing of leucyl-tRNALeu
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DOI：
10.1021/bi002915w
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发表时间：
2001-05-08
期刊：
影响因子：
2.9
通讯作者：
Martinis, SA
中科院分区：
文献类型：
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作者：
Mursinna, RS;Lincecum, TL;Martinis, SA
氨酰-tRNA合成酶通过准确地选择和激活用于正确tRNA的氨酰化的同源氨基酸来确保蛋白质合成的保真度。一些tRNA合成酶已经进化出与氨基酸活化位点分开的编辑活性位点，为氨基酸选择提供两个步骤或“筛”。这两种分子筛依赖于不同的氨基酸识别策略来显著提高氨酰化的准确性。我们已经进行了丙氨酸扫描诱变的大肠杆菌亮氨酰-tRNA合成酶的CP 1域，假设含有一个假定的编辑活性位点的保守的苏氨酸丰富的区域。纯化的突变体蛋白的表征导致鉴定出单一保守的苏氨酸，其防止关联亮氨酸氨基酸在tRNA的氨酰化后被水解。该苏氨酸突变为丙氨酸消除了编辑活性位点中同源氨基酸的区分。这提供了tRNA合成酶的编辑活性位点中的氨基酸识别机制的分子实例。
Aminoacyl-tRNA synthetases ensure the fidelity of protein synthesis by accurately selecting and activating cognate amino acids for aminoacylation of the correct tRNA. Some tRNA synthetases have evolved an editing active site that is separate from the amino acid activation site providing two steps or "sieves" for amino acid selection. These two sieves rely on different strategies for amino acid recognition to significantly enhance the accuracy of aminoacylation. We have performed alanine scanning mutagenesis in a conserved threonine-rich region of the Escherichia coli leucyl-tRNA synthetase's CP1 domain that is hypothesized to contain a putative editing active site. Characterization of purified mutant proteins led to the identification of a single conserved threonine that prevents the cognate leucine amino acid from being hydrolyzed after aminoacylation of the tRNA. Mutation of this threonine to an alanine eliminates discrimination of the cognate amino acid in the editing active site. This provides a molecular example of an amino acid discrimination mechanism in the tRNA synthetase's editing active site.