Structural basis for translational fidelity ensured by transfer RNA lysidine synthetase
Structural basis for translational fidelity ensured by transfer RNA lysidine synthetase
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DOI：
10.1038/nature08474
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发表时间：
2009-10-22
期刊：
影响因子：
64.8
通讯作者：
Nureki, Osamu
中科院分区：
文献类型：
--
作者：
Nakanishi, Kotaro;Bonnefond, Luc;Nureki, Osamu
前体转移RNA（pre-tRNA）的成熟包括切除5'前导序列和3'尾随序列、去除内含子和添加CCA末端（1-3）。在RNA分子采用适当的构象后，核苷酸修饰在tRNA加工的不同阶段被掺入。在细菌中，tRNA（Ile 2）赖氨酸合成酶（TilS）在tRNA（Ile 2）的第一个反密码子处将胞苷修饰为赖氨酸（L; 2-赖氨酰-胞苷）（参考文献4-9）。这种修饰将tRNA（Ile 2）从甲硫氨酸特异性tRNA转换为异亮氨酸特异性tRNA（9）。然而，在TilS修饰之前，甲硫氨酰-tRNA合成酶（MetRS）对tRNA（Ile 2）的氨酰化可能导致响应异亮氨酸密码子的甲硫氨酸的错误掺入。细菌用来避免这种陷阱的机制尚不清楚。在这里，我们表明，TilS酶特异性地识别和修改tRNA（Ile 2）在其前体形式，从而避免翻译错误。我们鉴定了从RNase-E缺陷型大肠杆菌中分离的前tRNA（Ile 2）中的赖氨酸修饰，并且没有检测到缺乏这种修饰的成熟tRNA（Ile 2）。我们的动力学分析表明，TilS可以修改这两种类型的RNA分子具有相当的效率。X射线晶体学和突变分析表明，TilS特异性地识别整个L形结构的前tRNA（Ile 2）通过广泛的相互作用加上连续的结构域运动。我们的研究结果证明了TilS如何阻止MetRS识别tRNA（Ile 2）并对其底物实现高特异性。这两个关键点形成了维持细菌中异亮氨酸密码子翻译保真度的基础。我们的研究结果也提供了一个理由，在tRNA生物合成过程中的前体水平纳入特定的修改的必要性。
Maturation of precursor transfer RNA (pre-tRNA) includes excision of the 5' leader and 3' trailer sequences, removal of introns and addition of the CCA terminus(1-3). Nucleotide modifications are incorporated at different stages of tRNA processing, after the RNA molecule adopts the proper conformation. In bacteria, tRNA(Ile2) lysidine synthetase (TilS) modifies cytidine into lysidine (L; 2-lysyl-cytidine) at the first anticodon of tRNA(Ile2) (refs 4-9). This modification switches tRNA(Ile2) from a methionine-specific to an isoleucine-specific tRNA(9). However, the aminoacylation of tRNA(Ile2) by methionyl-tRNA synthetase (MetRS), before the modification by TilS, might lead to the misincorporation of methionine in response to isoleucine codons. The mechanism used by bacteria to avoid this pitfall is unknown. Here we show that the TilS enzyme specifically recognizes and modifies tRNA(Ile2) in its precursor form, thereby avoiding translation errors. We identified the lysidine modification in pre-tRNA(Ile2) isolated from RNase-Edeficient Escherichia coli and did not detect mature tRNA(Ile2) lacking this modification. Our kinetic analyses revealed that TilS can modify both types of RNA molecule with comparable efficiencies. X-ray crystallography and mutational analyses revealed that TilS specifically recognizes the entire L-shape structure in pre-tRNA(Ile2) through extensive interactions coupled with sequential domain movements. Our results demonstrate how TilS prevents the recognition of tRNA(Ile2) by MetRS and achieves high specificity for its substrate. These two key points form the basis for maintaining the fidelity of isoleucine codon translation in bacteria. Our findings also provide a rationale for the necessity of incorporating specific modifications at the precursor level during tRNA biogenesis.