Regulation of Magnesium Homeostasis in Bacillus subtilis

描述（由申请人提供）：镁离子在所有活生物体中发挥许多重要作用，包括酶催化、结构完整性和微生物发病机制;然而，调节稳态的机制尚未完全了解。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，一个高度保守的金属传感RNA被广泛用于细菌中的转录后控制镁稳态。考虑到这种RNA元件的生物分布，它可能构成金属离子调节的共同模式，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的功能作为一个遗传传感器的镁在枯草芽孢杆菌，革兰氏阳性菌的模型系统，并控制表达的三个镁转运蛋白类之一。总之，这些数据表明，镁的缔合引起RNA内的构象转换，从而稳定转录终止信号以减少下游基因表达。基于这些观察结果，我们将阐明基于RNA的传感器识别镁的机制，并研究调控RNA对整体镁稳态控制的个体贡献。具体目标是：1）。阐明金属离子检测的结构基础。我们将完成金属结合RNA复合物的三维解析。使用其他生物物理和生物化学方法，我们将探索RNA-镁相互作用的特异性和化学计量，我们还将通过直接与镁配位的RNA基团的特异性取代进行功能测试。2）。我们将研究金属诱导的RNA折叠的动力学性质的凝胶和荧光为基础的技术与终止子的形成。我们还将调查是否不连续的转录延伸是专门与这些其他过程作为调控的最终要求。3）。我们预计，这种RNA介导的机制，虽然至关重要，可能只是镁稳态所需的多种遗传机制之一。我们将通过研究在其他镁调节基因的背景下候选转运基因的转录后调节来验证这一假设。

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.