Regulation of methionine metabolism in Bacillus subtilis

描述（申请人提供）：核糖开关rna在细菌中是一种重要的调控机制。这类rna由位于转录物前导区的复杂元件组成，位于调控编码序列的上游。这些RNA元件直接感知生理信号，诱导RNA发生结构变化，从而对下游基因表达产生影响。该项目将主要关注两类S-腺苷蛋氨酸（SAM）结合的核糖开关RNA， S盒和SMK盒，以及赖氨酸结合的L盒，硫胺素焦磷酸结合的Thi盒和响应温度变化的RNA热传感器。该项目的第一个主要目标是研究多种核糖开关rna识别特定配体的分子基础，以及S盒核糖开关自然变异中SAM敏感性差异的特征。这些努力还将包括利用获得的信息来设计新型的核开关，目的是测试这些分析的预测能力。第二个主要目标是研究在转录和翻译水平上运行的核糖体开关之间的结构和功能差异，以验证翻译核糖体开关（如SMK盒）在体内具有可逆操作的潜力的假设，允许在单个RNA转录物的生命周期内进行多个调控决策。这些研究将包括详细分析SMK盒RNA的无配体形式以及无配体和配体结合形式之间的转变。第三个主要目标是分析核糖体开关元件与其他调控机制之间的相互作用，以枯草芽孢杆菌metK基因编码SAM合成酶为例。总体而言，该项目将提供基于rna的新型基因调控机制的基本信息，并将为利用这些机制的病原生物的代谢调控提供见解。革兰氏阳性病原体通常使用与枯草芽孢杆菌密切相关的调节机制，而枯草芽孢杆菌是这项工作的模式生物。致病性决定因素的表达通常受到生理信号的调节，了解细胞如何监测这些信号对于理解细菌的毒力很重要。也有可能许多新的类似核糖体开关的机制仍未被发现，而提出的工作将为研究这些机制和预测它们在细胞内的功能提供重要的工具。

DESCRIPTION (provided by applicant): Riboswitch RNAs represent an important regulatory mechanism in bacteria. RNAs of this type consist of complex elements positioned in the leader region of a transcript, upstream of the regulated coding sequence(s). These RNA elements directly sense a physiological signal that induces a structural change in the RNA, resulting in an effect on downstream gene expression. This project will focus primarily on two classes of S-adenosylmethionine (SAM)-binding riboswitch RNAs, the S box and the SMK box, with additional efforts on the lysine-binding L box, the thiamine pyrophosphate-binding Thi box, and RNA thermosensors that respond to changes in temperature. The first major goal of the project is to investigate the molecular basis for specific ligand recognition by multiple riboswitch RNAs, and the features responsible for differential SAM sensitivity in natural variants of the S box riboswitch. These efforts will also include using the information obtained to engineer novel classes of riboswitches with the goal of testing the predictive power of these analyses. The second major goal is to investigate the structural and functional differences between riboswitches that operate at the transcriptional and translational levels, to test the hypothesis that translational riboswitches (like the SMK box) have the potential to operate reversibly in vivo, allowing multiple regulatory decisions within the lifetime of a single RNA transcript. These studies will include detailed analysis of the ligand-free form of the SMK box RNA and the transition between the ligand-free and ligand-bound forms. The third major goal involves analysis of the interplay between riboswitch elements and other regulatory mechanisms, using the Bacillus subtilis metK gene, encoding SAM synthetase, as an example. Overall, this project will provide basic information about novel RNA-based mechanisms of gene regulation, and will also provide insight into metabolic regulation in pathogenic organisms that use these mechanisms. Gram- positive pathogens generally use regulatory mechanisms closely related to those found in Bacillus subtilis, the model organism for this work. Expression of determinants for pathogenicity are often regulated in response to physiological signals, and understanding how the cell monitors these signals is important for understanding bacterial virulence. It is also likely that many new riboswitch-like mechanisms remain to be uncovered, and the proposed work will provide important tools for investigation of these mechanisms, and predicting how they function within the cell. PUBLIC HEALTH RELEVANCE: RNA-mediated regulation has recently emerged as a central player in all organisms. This study is directed toward the analysis of metabolite-binding riboswitches, a class of regulatory RNAs that directly sense a physiological signal and transmit that information to the gene expression machinery via an RNA structural rearrangement. This mechanism is widely used in bacteria, including in a number of important pathogens, and has also been identified in archaea and eukaryotes. The goal of this project is to investigate the molecular mechanisms underlying RNA-mediated ligand recognition and gene regulation in response to modulation of RNA structure.