Regulation of Magnesium Homeostasis in Bacillus subtilis

描述(申请人提供)：镁离子在所有生物体中发挥许多基本作用，包括酶催化、结构完整性和微生物致病；然而，调节体内平衡的机制尚不完全清楚。蛋白质通常被认为是金属离子的哨兵；然而，我们的中心假设是，高度保守的金属感应RNA在细菌中被广泛用于转录后控制镁的动态平衡。鉴于这种RNA元素的生物分布，它很可能构成一种常见的金属离子调节模式，它的发现表明存在其他基于RNA的金属离子传感器。我们已经证明，这种RNA在枯草芽孢杆菌中作为镁的遗传传感器发挥作用，并控制三种镁转运蛋白之一的表达。综上所述，这些数据表明，镁的结合激发了RNA中的构象转换，进而稳定了转录终止信号，从而减少了下游基因的表达。在这些观察的基础上，我们将阐明基于RNA的传感器识别镁的机制，并调查调控RNA对整体镁稳态控制的个体贡献。具体目标是：1)。阐明了金属离子检测的结构基础。我们将完成金属结合的RNA复合体的三维解析。使用其他生物物理和生化方法，我们将探索RNA-镁相互作用的特异性和化学计量比，我们还将通过直接与镁配位的RNA基团的特定取代来进行功能测试。2)。我们将用凝胶和荧光技术研究金属诱导的RNA折叠的动力学性质，与终止子的形成有关。我们还将调查不连续的转录延长是否特定地与这些其他过程结合在一起，作为调控的最终要求。3)。我们预计，这种由RNA介导的机制虽然至关重要，但可能只是镁稳态所需的多种遗传机制之一。我们将在其他镁调控基因的背景下，通过研究候选运输基因的转录后调控来检验这一假设。

DESCRIPTION (provided by applicant): Magnesium ions perform many essential roles in all living organisms, including enzymatic catalysis, structural integrity, and microbial pathogenesis; however, the mechanisms that regulate homeostasis are incompletely understood. Proteins are generally assumed to be the sentinels for metal ions; however, our central hypothesis is that a highly conserved metal-sensing RNA is broadly used among bacteria for posttranscriptional control of magnesium homeostasis. Given the biological distribution of this RNA element it is likely to constitute a common mode of metal ion regulation and its discovery suggests the presence of other RNA-based metal ion sensors. We have demonstrated that this RNA functions as a genetic sensor for magnesium in Bacillus subtilis, the model system for Gram-positive bacteria, and controls expression of one of the three magnesium transporter classes. Together, these data suggest that association of magnesium provokes a conformational switch within the RNA that in turn stabilizes a transcription termination signal for reduction of downstream gene expression. Based on these observations we will elucidate the mechanism of magnesium recognition by the RNA-based sensor and investigate the individual contribution of the regulatory RNA to overall magnesium homeostasis control. The Specific Aims are to: 1). Elucidate the structural basis for metal ion detection. We will complete the three-dimensional resolution of the metal-bound RNA complex. Using additional biophysical and biochemical methods we will explore the specificity and stoichiometry of RNA-magnesium interactions, which we will also functionally test through specific substitution of RNA groups that directly coordinate to magnesium. 2). We will study the dynamical nature of metal-induced RNA folding by gel- and fluorescence-based techniques in relation to terminator formation. We will also investigate whether discontinuous transcriptional elongation is specifically coupled with these other processes as a final requirement for regulation. 3). We expect that this RNA-mediated mechanism, although vitally important, may only be one of multiple genetic mechanisms required for magnesium homeostasis. We will test this hypothesis by investigating posttranscriptional regulation of the candidate transport genes within the context of additional magnesium- regulated genes.