State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, Shanghai 200031, China.
Nucleic Acids Res. 2011 Nov 1;39(20):8857-68. doi: 10.1093/nar/gkr595. Epub 2011 Jul 20.
Aminoacyl-tRNA synthetases (aaRSs) are remarkable enzymes that are in charge of the accurate recognition and ligation of amino acids and tRNA molecules. The greatest difficulty in accurate aminoacylation appears to be in discriminating between highly similar amino acids. To reduce mischarging of tRNAs by non-cognate amino acids, aaRSs have evolved an editing activity in a second active site to cleave the incorrect aminoacyl-tRNAs. Editing occurs after translocation of the aminoacyl-CCA₇₆ end to the editing site, switching between a hairpin and a helical conformation for aminoacylation and editing. Here, we studied the consequence of nucleotide changes in the CCA₇₆ accepting end of tRNA(Leu) during the aminoacylation and editing reactions. The analysis showed that the terminal A₇₆ is essential for both reactions, suggesting that critical interactions occur in the two catalytic sites. Substitutions of C₇₄ and C₇₅ selectively decreased aminoacylation keeping nearly unaffected editing. These mutations might favor the regular helical conformation required to reach the editing site. Mutating the editing domain residues that contribute to CCA₇₆ binding reduced the aminoacylation fidelity leading to cell-toxicity in the presence of non-cognate amino acids. Collectively, the data show how protein synthesis quality is controlled by the CCA₇₆ homogeneity of tRNAs.
氨酰-tRNA 合成酶（aaRSs）是一种非凡的酶，负责精确识别和连接氨基酸和 tRNA 分子。准确氨酰化的最大困难似乎在于区分高度相似的氨基酸。为了减少非对应氨基酸对 tRNA 的错误 charging，aaRSs 在第二个活性位点中进化出一种编辑活性，以切割不正确的氨酰-tRNA。编辑发生在氨酰-CCA₇₆末端转移到编辑位点之后，在氨酰化和编辑过程中切换发夹和螺旋构象。在这里，我们研究了在氨酰化和编辑反应过程中 tRNA(Leu)的 CCA₇₆ 接受末端的核苷酸变化的后果。分析表明，末端 A₇₆对于这两个反应都是必不可少的，这表明在两个催化位点中发生了关键相互作用。C₇₄ 和 C₇₅ 的取代选择性地降低了氨酰化，而编辑几乎不受影响。这些突变可能有利于达到编辑位点所需的规则螺旋构象。突变与 CCA₇₆ 结合有关的编辑结构域残基降低了氨酰化保真度，导致在存在非对应氨基酸时细胞毒性。总的来说，这些数据表明蛋白质合成质量如何受到 tRNAs 的 CCA₇₆ 同质性的控制。
