Regulation of Magnesium Homeostasis in Bacillus subtilis

枯草芽孢杆菌中镁稳态的调节

• 批准号: 7489391
• 负责人: Wade Winkler
• 金额: $ 23.86万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-27 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7489391
• 关键词: 5' Untranslated Regions; Bacillus subtilis; Bacteria; Binding; Biochemical; Biological; Biological Models; Biological Process; Carrier Proteins; Catalysis; Class; Complex; Coupled; Data; Defect; Detection; Elements; Fluorescence; Fluorescence Spectroscopy; Gel; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Gram-Positive Bacteria; Helix (Snails); Homeostasis; Hydroxyl Radical; Individual; Investigation; Ions; Kinetics; Life; Magnesium; Measurement; Mediating; Metals; Methodology; Methods; Molecular; Nature; Nucleic Acid Regulatory Sequences; Organism; Pathogenesis; Pathway interactions; Process; Proteins; Purpose; RNA; RNA Binding; RNA Folding; Recording of previous events; Regulation; Relative (related person); Reporter; Research; Research Personnel; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Role; Sentinel; Signal Transduction; Specificity; Structure; Techniques; Testing; Thermodynamics; Time; Transcript; attenuation; base; ear helix; interest; magnesium ion; microbial; nucleotide analog; programs; research study; response; sedimentation velocity; sensor; stoichiometry; three-dimensional modeling; transcription termination

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.

描述（由申请人提供）：镁离子在所有生物体中都起着许多基本作用，包括酶促催化，结构完整性和微生物发病机理；但是，调节体内平衡的机制尚不完全理解。通常认为蛋白质是金属离子的前哨。但是，我们的中心假设是，在细菌中广泛使用了高度保守的金属敏感性RNA，用于对镁稳态的转录后控制。鉴于该RNA元件的生物学分布可能构成金属离子调节的常见模式，并且其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，该RNA充当枯草芽孢杆菌（革兰氏阳性细菌的模型系统）中镁的遗传传感器，并控制了三种镁转运蛋白类别之一的表达。总之，这些数据表明，镁的关联会挑衅RNA内的构象开关，进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，我们将通过基于RNA的传感器阐明镁识别的机制，并研究调节RNA对总体镁稳态控制的个体贡献。具体目的是：1）。阐明金属离子检测的结构基础。我们将完成金属结合的RNA复合物的三维分辨率。使用其他生物物理和生化方法，我们将探讨RNA-Magnesium相互作用的特异性和化学计量，我们还将通过特定的RNA基团进行功能​​测试，RNA基团直接与镁配位。 2）。我们将研究金属诱导的RNA折叠的动力学性质，而基于荧光的技术与终结剂形成有关。我们还将调查不连续的转录伸长是否专门与其他过程相结合，作为调节的最终要求。 3）。我们预计这种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。我们将通过在其他镁调节基因的背景下研究候选转运基因的转录后调节来检验这一假设。