Regulation of Magnesium Homeostasis in Bacillus subtilis

枯草芽孢杆菌中镁稳态的调节

• 批准号: 7665041
• 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/7665041
• 关键词: 5' Untranslated Regions; Bacillus subtilis; Bacteria; Binding; Biochemical; Biological; Biological Models; Biological Process; Carrier Proteins; Catalysis; Complex; Coupled; Data; Defect; Detection; Elements; Fluorescence; Fluorescence Spectroscopy; Gel; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Gram-Positive Bacteria; 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; 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; 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在细菌中广泛用于镁稳态的转录后控制。考虑到这种RNA元件的生物分布，它很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在革兰氏阳性菌的模型系统枯草芽孢杆菌中作为镁的遗传传感器起作用，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内部的构象开关，从而稳定了转录终止信号，从而减少了下游基因的表达。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，并研究调节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。使用额外的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学，我们也将通过直接与镁协调的RNA基团的特异性替代进行功能测试。2)。我们将通过凝胶和荧光技术研究金属诱导RNA折叠的动力学性质，这些技术与终止体的形成有关。我们还将研究不连续转录延伸是否与这些其他过程特异性耦合，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，但可能只是镁稳态所需的多种遗传机制之一。我们将通过研究候选转运基因在其他镁调控基因背景下的转录后调控来验证这一假设。