Structural transitions induced by the interaction between tRNAGly and the Bacillus subtilis glyQS T box leader RNA
Structural transitions induced by the interaction between tRNAGly and the Bacillus subtilis glyQS T box leader RNA
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DOI：
10.1016/j.jmb.2005.03.061
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发表时间：
2005-06-03
影响因子：
5.6
通讯作者：
Henkin, TM
中科院分区：
文献类型：
--
作者：
Yousef, MR;Grundy, FJ;Henkin, TM
T盒系统通过提前终止转录来调节革兰氏阳性菌中氨基酸相关基因的表达。全长 mRNA 的合成需要通过同源不带电 tRNA 稳定 5' 未翻译前导 RNA 中的抗终止子元件。枯草芽孢杆菌 glyQS 基因（编码甘氨酰-tRNA 合成酶）的 tRNA (Gly) 依赖性抗终止可以在纯化的体外转录系统中重现，表明新生转录物足以与 tRNA 相互作用。之前的遗传分析证明了前导 RNA 中单个密码子与 tRNA 反密码子以及反终止子和 tRNA 受体末端之间的碱基配对。在这项研究中，我们建立了 tRNA(Gly) 与噬菌体 T7 RNA 聚合酶产生的 glyQS 前导 RNA 特异性结合的条件。结构图研究揭示了 tRNA (Gly) 诱导的 glyQS 前导 RNA 中与 tRNA 相互作用的两个已知位点以及这些位点之间的其他区域的保护。直接证明了前导 RNA 的竞争性终止子和反终止子形式之间的 tRNA 依赖性结构转换。在反密码子环中检测到与 glyQS 前导 RNA 结合后 tRNA(Gly) 的变化，这与与指定序列的配对一致，并且在 D 环中高度保守的 G19 中检测到，类似于核糖体中密码子-反密码子相互作用诱导的效应。这项研究为 tRNA(Gly) 与全长体外转录的 glyQS 先导 RNA 直接相互作用提供了生化证据，并初步了解了复合物内两种 RNA 伴侣的结构调节。 (c) 2005 Elsevier Ltd. 保留所有权利。
The T box system regulates expression of amino acid-related genes in Gram-positive bacteria through premature termination of transcription. Synthesis of the full-length mRNA requires stabilization of an antiterminator element in the 5' untranslated leader RNA by the cognate uncharged tRNA. tRNA (Gly)-dependent antitermination of the Bacillus subtilis glyQS gene (encoding glycyl-tRNA synthetase) can be reproduced in a purified in vitro transcription system, indicating that the nascent transcript is sufficient for interaction with the tRNA. Genetic analyses previously demonstrated base pairing of a single codon in the leader RNA with the tRNA anticodon, and between the antiterminator and the tRNA acceptor end. In this study, we established conditions for specific binding of tRNA(Gly) to glyQS leader RNA generated by phage T7 RNA polymerase. Structural mapping studies revealed tRNA (Gly)-induced protection in the glyQS leader RNA at the two known sites of interaction with the tRNA, as well as at other regions between these sites. The proposed tRNA-dependent structural switch between the competing terminator and antiterminator forms of the leader RNA was demonstrated directly. Changes in tRNA(Gly) upon binding to glyQS leader RNA were detected in the anticodon loop, consistent with pairing with the specifier sequence, and in the highly conserved G19 in the D-loop, similar to effects induced by codon-anticodon interaction in the ribosome. This study provides biochemical evidence for direct interaction of tRNA(Gly) with full-length in vitro transcribed glyQS leader RNA, and an initial view of structural modulations of both RNA partners within the complex. (c) 2005 Elsevier Ltd. All rights reserved.