Regulation of methionine metabolism in Bacillus subtilis

描述(申请人提供)：核糖开关RNA代表细菌中的一种重要的调节机制。这种类型的RNA由位于转录前导区的复杂元件组成，位于受调控的编码序列的上游(S)。这些RNA元件直接感应生理信号，引起RNA的结构变化，从而影响下游基因的表达。该项目将主要关注两类与S-腺苷蛋氨酸结合的核糖开关RNA，即S盒和SMK盒，以及与赖氨酸结合的L盒、与硫胺素焦磷酸结合的THI盒以及响应温度变化的RNA温度传感器。该项目的第一个主要目标是研究多个核糖开关RNA识别特定配体的分子基础，以及导致S盒核糖开关自然变异体对SAM的不同敏感性的特征。这些努力还将包括利用获得的信息设计新型核糖开关，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工作的核糖开关之间的结构和功能差异，测试翻译核糖开关(如SMK盒)具有在体内可逆工作的潜力的假设，允许在单个RNA转录本的生命周期内做出多个调控决定。这些研究将包括对SMK盒RNA的无配体形式以及无配体形式和配体结合形式之间的转换进行详细分析。第三个主要目标涉及分析核糖开关元件和其他调节机制之间的相互作用，以编码SAM合成酶的枯草杆菌metK基因为例。总体而言，该项目将提供关于基于RNA的新的基因调控机制的基本信息，并将提供对使用这些机制的病原体的代谢调控的洞察。革兰氏阳性病原体通常使用与枯草杆菌密切相关的调节机制，枯草杆菌是这项工作的模式生物。致病性决定因素的表达经常受到生理信号的调节，了解细胞如何监控这些信号对于了解细菌的毒力很重要。也有可能许多新的核糖开关机制仍未被发现，拟议的工作将为研究这些机制并预测它们在细胞内的功能提供重要工具。 与公共健康相关：最近，RNA介导的调控在所有生物体中都扮演着核心角色。这项研究旨在分析代谢物结合核糖开关，这是一类直接感知生理信号并通过RNA结构重排将信息传递给基因表达机制的调控RNA。这一机制被广泛应用于细菌，包括一些重要的病原体，也已在古生物和真核生物中被发现。本项目的目标是研究RNA介导的配体识别和基因调控响应RNA结构调整的分子机制。

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.