Anticodon recognition in evolution - Switching tRNA specificity of an aminoacyl-tRNA synthetase by site-directed peptide transplantation
Anticodon recognition in evolution - Switching tRNA specificity of an aminoacyl-tRNA synthetase by site-directed peptide transplantation
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DOI：
10.1074/jbc.m302618200
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发表时间：
2003-08-15
影响因子：
4.8
通讯作者：
Plateau, P
中科院分区：
文献类型：
--
作者：
Brevet, A;Chen, J;Plateau, P
高度保守的天冬酰胺酰-、天冬酰胺酰-和赖氨酰-tRNA合成酶组成氨酰-tRNA合成酶的一个亚类，称为IIb。这三种酶在其N末端具有OB-折叠延伸。该延伸的功能是特异性识别tRNA的反密码子三联体。细菌内酰-和赖氨酰-tRNA合成酶与tRNA复合的三维模型表明，沿着OB折叠的五条β链的氨基酸残基沿着的刚性支架容纳反密码子中心的碱基U。相邻反密码子碱基的结合通过与连接链4和5的柔性环（L（45））的相互作用发生。结果，可以尝试通过将赖氨酰-tRNA合成酶的小环L（45）移植到赖氨酰-tRNA合成酶内，将赖氨酰- tRNA合成酶的特异性从tRNA（Lys）（反密码子UUU）转换为tRNA（Asp）（GUC）。在这种移植后，赖氨酰-tRNA合成酶失去其氨酰化tRNA（Lys）的能力。作为交换，嵌合酶获得了用赖氨酸装载tRNA（Asp）的能力。在给予tRNA（Asp）底物tRNA（Lys）的双碱基后，特异性转变得到改善。还在体内建立了特异性的变化。事实上，移植的赖氨酰-tRNA合成酶成功地抑制了插入β-内酰胺酶基因中的错义Lys -> Asp突变。这些结果在功能上确立了IIb类氨酰-tRNA合成酶的小肽区域中的序列变异有助于核酸识别的规范。因为这个肽元件不是核心催化结构的一部分，所以它可能独立于这些合成酶的活性位点而进化。
The highly conserved aspartyl-, asparaginyl-, and lysyl-tRNA synthetases compose one subclass of amino-acyl-tRNA synthetases, called IIb. The three enzymes possess an OB-folded extension at their N terminus. The function of this extension is to specifically recognize the anticodon triplet of the tRNA. Three-dimensional models of bacterial aspartyl- and lysyl-tRNA synthetases complexed to tRNA indicate that a rigid scaffold of amino acid residues along the five beta-strands of the OB-fold accommodates the base U at the center of the anticodon. The binding of the adjacent anticodon bases occurs through interactions with a flexible loop joining strands 4 and 5 (L(45)). As a result, a switching of the specificity of lysyl-tRNA synthetase from tRNA(Lys) (anticodon UUU) toward tRNA(Asp) (GUC) could be attempted by transplanting the small loop L(45) of aspartyl- tRNA synthetase inside lysyl-tRNA synthetase. Upon this transplantation, lysyl-tRNA synthetase loses its capacity to aminoacylate tRNA(Lys). In exchange, the chimeric enzyme acquires the capacity to charge tRNA(Asp) with lysine. Upon giving the tRNA(Asp) substrate the discriminator base of tRNA(Lys), the specificity shift is improved. The change of specificity was also established in vivo. Indeed, the transplanted lysyl-tRNA synthetase succeeds in suppressing a missense Lys --> Asp mutation inserted into the beta-lactamase gene. These results functionally establish that sequence variation in a small peptide region of subclass IIb aminoacyl-tRNA synthetases contributes to specification of nucleic acid recognition. Because this peptide element is not part of the core catalytic structure, it may have evolved independently of the active sites of these synthetases.