Domain-domain communication for tRNA aminoacylation: The importance of covalent connectivity
Domain-domain communication for tRNA aminoacylation: The importance of covalent connectivity
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DOI：
10.1021/bi050285y
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发表时间：
2005-05-17
期刊：
影响因子：
2.9
通讯作者：
Hou, YM
中科院分区：
文献类型：
--
作者：
Zhang, CM;Hou, YM
氨酰-tRNA合成酶与tRNA形成复合物以催化活化的氨基酸转移到tRNA的3'末端。tRNA合成酶复合物大致分为合成酶的激活域和tRNA结合域，它们分别与tRNA的受体和反密码子末端相互作用。通过大肠杆菌半胱氨酰-tRNA合成酶（CysRS）对tRNA进行有效的氨酰化需要两个结构域，尽管用于长距离结构域-结构域通信的途径还不很清楚。以往的研究表明，切割成受体和反密码子螺旋的tRNA（Cys）严重降低了氨酰化的效率，这表明通讯需要共价连续的tRNA骨架。在这里，我们测试了通信是否需要合成酶骨架的连续性。两个N端片段和一个C端片段。coli CysRS。虽然N-末端片段在腺苷酸合成中具有活性，但它们在tRNA氨酰化的催化效率和特异性方面存在严重缺陷。相反，虽然C-末端片段没有催化活性，但它能够结合并区分tRNA。然而，将C-末端片段反式添加到N-末端片段并没有将N-末端片段的氨酰化效率提高到全长酶的水平。这些结果强调了CysRS和tRNA（Cys）的共价连续性对于有效的tRNA氨酰化的重要性，并突出了限制tRNA合成酶复合物用于结构域-结构域通信的能量成本。重要的是，这项研究还提供了新的见解存在的几个天然的“分裂”合成酶，现在确定从基因组测序项目。
Aminoacyl-tRNA synthetases form complexes with tRNA to catalyze transfer of activated amino acids to the 3' end of tRNA. The tRNA synthetase complexes are roughly divided into the activation and tRNA-binding domains of synthetases, which interact with the acceptor and anticodon ends of tRNAs, respectively. Efficient aminoacylation of tRNA by Escherichia coli cysteinyl-tRNA synthetase (CysRS) requires both domains, although the pathways for the long-range domain-domain communication are not well understood. Previous studies show that dissection of tRNA(Cys) into acceptor and anticodon helices seriously reduces the efficiency of aminoacylation, suggesting that communication requires covalent continuity of the tRNA backbone. Here we tested if communication requires the continuity of the synthetase backbone. Two N-terminal fragments and one C-terminal fragment of E. coli CysRS were generated. While the N-terminal fragments were active in adenylate synthesis, they were severely defective in the catalytic efficiency and specificity of tRNA aminoacylation. Conversely, although the C-terminal fragment was not catalytically active, it was able to bind and discriminate tRNA. However, addition of the C-terminal fragment to an N-terminal fragment in trans did not improve the aminoacylation efficiency of the N-terminal fragment to the level of the full-length enzyme. These results emphasize the importance of covalent continuity of both CysRS and tRNA(Cys) for efficient tRNA aminoacylation, and highlight the energetic costs of constraining the tRNA synthetase complex for domain-domain communication. Importantly, this study also provides new insights into the existence of several natural "split" synthetases that are now identified from genomic sequencing projects.