Regulation of Bacillus subtilis tRNA synthetase genes

描述（申请人提供）：在枯草芽孢杆菌和相关生物中，许多涉及氨基酸代谢的基因都受T box机制的调控，T box机制是一种新的转录终止控制系统。该家族中每个基因的表达依赖于一个特定的未带电tRNA与新生RNA转录物的结合，从而导致一个终止信号的读取，否则会减弱下游基因的表达。tRNA识别需要tRNA反密码子与先导RNA中的单个密码子配对，即“指示子序列”，以及tRNA的受体端与与终止子螺旋形成竞争的反终止子元件配对；这些配对对于反终止是必要的，但不是充分的。tRNAGly依赖的枯草芽孢杆菌glyQS基因抗体可以在纯化的系统中复制，并且tRNAGly与glyQS先导RNA的结合导致整个RNA的结构变化。T盒rna直接特异性识别同源效应分子调控基因表达的能力使这些rna成为调控rna的核糖体开关家族。下一个项目周期将针对tRNA依赖性抗体的分子机制进行详细分析，以及T盒rna特异性识别tRNA。我们将重点关注与已知碱基配对区域不同的RNA元件，以及glyQS模型RNA（代表自然缺失变体）与更常见的包含附加结构元件（功能未知）的复杂T盒RNA之间的功能差异。我们还将研究在转录终止水平（主要在厚壁菌门中发现）和在翻译起始水平（主要在放线菌中发现，包括分枝杆菌）操作的T盒元件之间的机制差异。进一步表征T盒机制对于开发针对该机制的新型抗菌药物的长期目标至关重要，该机制存在于许多革兰氏阳性病原体中。近年来，RNA作为调控分子的作用越来越受到重视，T盒系统代表了RNA介导基因调控的独特分子机制，是tRNA在细胞中的新功能。公共卫生相关性：rna介导的调控最近在所有生物体中都发挥了核心作用。本研究旨在分析T盒系统，这是一种独特的调节机制，RNA转录物直接感知特定的tRNA分子来控制基因表达。T盒机制广泛用于革兰氏阳性菌（包括重要病原体），以调节多种必需基因，这项工作的长期目标是鉴定出使该系统失活的新型抗菌药物，从而阻止其生长。

DESCRIPTION (provided by applicant): Many genes involved in amino acid metabolism in Bacillus subtilis and related organisms are regulated by the T box mechanism, a novel transcription termination control system. Expression of each gene in this family depends on binding of a specific uncharged tRNA to the nascent RNA transcript, resulting in readthrough of a termination signal that otherwise attenuates downstream gene expression. tRNA recognition requires pairing of the tRNA anticodon with a single codon, the "Specifier Sequence," in the leader RNA, as well as pairing of the acceptor end of the tRNA to an antiterminator element that competes with formation of the terminator helix; these pairing are necessary but not sufficient for antitermination. tRNAGly-dependent antitermination of the B. subtilis glyQS gene can be reproduced in a purified system, and binding of tRNAGly to glyQS leader RNA results in structural changes throughout the RNA. The ability of T box RNAs to directly and specifically recognize their cognate effector molecule to regulate gene expression places these RNAs in the riboswitch family of regulatory RNAs. The next project period will be directed toward detailed analysis of the molecular mechanism of tRNA-dependent antitermination, and specific tRNA recognition by T box RNAs. We will focus on RNA elements distinct from the known regions of base-pairing, and functional differences between the glyQS model RNA, which represents a natural deletion variant, and the more common complex T box RNAs that contain additional structural elements, the function of which is unknown. We will also investigate the mechanistic difference between T box elements that operate at the level of transcription termination (found primarily in Firmicutes), and those that operate at the level of translation initiation (found primarily in Actinomycetes, including Mycobacterium sp.). Further characterization of the T box mechanism is essential to the long-term goal of developing novel antimicrobial agents that target this mechanism, which is found in many Gram-positive pathogens. The role of RNA as a regulatory molecule has been increasingly appreciated in recent years, and the T box system represents a unique molecular mechanism for RNA-mediated gene regulation, and a new function for tRNA in the cell. PUBLIC HEALTH RELEVANCE: RNA-mediated regulation has recently emerged as a central player in all organisms. This study is directed toward analysis of the T box system, a unique regulatory mechanism in which RNA transcripts directly sense a specific tRNA molecule to control gene expression. The T box mechanism is widely used in Gram-positive bacteria, including important pathogens, to regulate multiple essential genes, and a long-term goal of this work is the identification of novel antimicrobial agents that inactivate this system, therefore preventing growth.