tRNA t6A 的结构-功能分析--Aquifex aeolicus TsaD2B2 四聚体与 TsaE 复合物的催化、组装和热稳定性。

IF 4 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Shuze Lu, Mengqi Jin, Zhijiang Yu, Wenhua Zhang
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引用次数: 0

摘要

位于 tRNA 第 37 位的通用 N6-苏氨酰氨基甲酰腺苷(t6A)是促进翻译保真度所需的核心转录后修饰之一。在细菌中,TsaC 利用 L-苏氨酸、碳酸氢盐和 ATP 生成中间体苏氨酰氨基甲酰基腺苷酸(TC-AMP),其中的 TC-分子在 TsaB 和 TsaE 的支持下由 TsaD 转移到 tRNA A37 的 N6 原子上生成 t6A。TsaD 和 TsaB 形成一个 TsaDB 二聚体,tRNA 和 TsaE 与之竞争结合。TsaD 的催化机理以及 TsaB 和 TsaE 的辅助作用仍有待全面阐明。在本研究中,我们利用嗜热水蚤的重组 TsaC、TsaD-TsaB 和 TsaE 重建了 tRNA t6A 的生物合成,并测定了 TsaD2B2 四聚体的 apo 形式和 ADP 结合形式的晶体结构。我们的 TsaD2B2-TsaE-tRNA 模型耦合功能验证表明,tRNA 或 TsaE 与 TsaDB 的结合受 TsaB 的 C 端尾和 TsaD 的螺旋发夹 α1-α2 的调节。A. aeolicus 的 TsaD2B2 或 TsaDB 具有依赖于二价离子的基础 t6A 催化活性,这种活性在 TsaE 的刺激下以 ATP 消耗为代价。我们的数据表明,TsaE 与 TsaDB 结合会诱导 TsaD 的 α1、α2、α6、α7 和 α8 以及 TsaB 的 C 端尾部发生构象变化,从而导致 tRNA t6A 和 AMP 的释放。ATP 水解驱动的 TsaE 与 TsaDB 的解离重置了 TsaDB 的活性构象。嗜热 TsaDB 的二聚化提高了热稳定性,促进了 TsaD2B2-tRNA 的 t6A 催化活性,其中反密码子茎中的 GC 碱基对是嗜热 tRNA 在较高温度下正确折叠所必需的。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Structure-function analysis of tRNA t6A-catalysis, assembly and thermostability of Aquifex aeolicus TsaD2B2 tetramer in complex with TsaE.

The universal N6-threonylcarbamoyladenosine (t6A) at position 37 of tRNAs is one of core post-transcriptional modifications that are needed for promoting translational fidelity. In bacteria, TsaC utilizes L-threonine, bicarbonate and ATP to generate an intermediate threonylcarbamoyladenylate (TC-AMP), of which the TC-moiety is transferred to N6 atom of tRNA A37 to generate t6A by TsaD with support of TsaB and TsaE. TsaD and TsaB form a TsaDB dimer to which tRNA and TsaE are competitively bound. The catalytic mechanism of TsaD and auxiliary roles of TsaB and TsaE remain to be fully elucidated. In this study, we reconstituted tRNA t6A biosynthesis using recombinant TsaC, TsaD-TsaB and TsaE from thermophilic Aquifex aeolicus and determined crystal structures of apo-form and ADP-bound form of TsaD2B2 tetramer. Our TsaD2B2-TsaE-tRNA model coupled functional validations reveal that the binding of tRNA or TsaE to TsaDB is regulated by C-terminal tail of TsaB and a helical hairpin α1-α2 of TsaD. A. aeolicus TsaD2B2 or TsaDB possesses a basal divalent ion-dependent t6A-catalytic activity that is stimulated by TsaE at the cost of ATP consumption. Our data suggest that binding of TsaE to TsaDB induces conformational changes of α1, α2, α6, α7 and α8 of TsaD and C-terminal tail of TsaB, leading to release of tRNA t6A and AMP. ATP hydrolysis-driven dissociation of TsaE from TsaDB resets an active conformation of TsaDB. Dimerization of thermophilic TsaDB enhances thermostability and promotes t6A-catalytic activity of TsaD2B2-tRNA, of which GC base pairs in anticodon stem are needed for correct folding of thermophilic tRNA at higher temperatures.

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来源期刊
Journal of Biological Chemistry
Journal of Biological Chemistry Biochemistry, Genetics and Molecular Biology-Biochemistry
自引率
4.20%
发文量
1233
期刊介绍: The Journal of Biological Chemistry welcomes high-quality science that seeks to elucidate the molecular and cellular basis of biological processes. Papers published in JBC can therefore fall under the umbrellas of not only biological chemistry, chemical biology, or biochemistry, but also allied disciplines such as biophysics, systems biology, RNA biology, immunology, microbiology, neurobiology, epigenetics, computational biology, ’omics, and many more. The outcome of our focus on papers that contribute novel and important mechanistic insights, rather than on a particular topic area, is that JBC is truly a melting pot for scientists across disciplines. In addition, JBC welcomes papers that describe methods that will help scientists push their biochemical inquiries forward and resources that will be of use to the research community.
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