REGULATION OF THE TRYTOPHAN GENES IN BACILLUS

芽孢杆菌中色氨酸基因的调控

• 批准号: 6387307
• 负责人: PAUL D GOLLNICK
• 金额: $ 20.79万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-15 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6387307
• 关键词: Bacillus; RNA binding protein; bacterial RNA; bacterial genetics; genetic transcription; protein binding; tryptophan

Controlling transcription termination prior to the coding region is a commonly used strategy to regulate gene expression in bacteria, including many with importance to human health. Such control mechanisms are collectively termed attenuation and antitermination. The proposed research will investigate the mechanisms by which RNA binding proteins recognize and bind to specific sites in RNA, and how these interactions regulate transcription attenuation. The model system of study is the TRAP protein (trp RNA-binding Attenuation Protein), an RNA binding protein that regulates transcription attenuation of the tryptophan biosynthetic genes in Bacillus subtilis and related Bacilli. In the presence of excess tryptophan, TRAP is activated to bind to a series of 11 GAG or UAG repeats in the 5' leader region of the trp operon. This binding induces formation of a transcription terminator, which halts expression of the genes. TRAP is an 11 subunit protein that forms a symmetric ring. RNA binds to TRAP by wrapping around the outer perimeter of the protein ring. The detailed mechanism by which TRAP associates with its RNA target will be characterized using a combination of equilibrium binding studies, nucleoside analogs, and rapid-quench stopped-flow studies. TRAP is activated to bind RNA by binding 11 molecules of L-tryptophan. Crystallography, genetics and biochemical approaches will be used to determine the mechanism by which tryptophan binding activates TRAP. The third objective is to develop a more detailed understanding of the mechanism of TRAP mediated transcription attenuation. Key elements of the TRAP binding site in the trp leader will be altered and the effects of these changes on attenuation studied in vivo using a trpE''-'lacZ gene fusion. These studies will be guided by the information learned from in vitro studies of the TRAP/RNA interaction.

在编码区之前控制转录终止是 调节细菌基因表达的常用策略，包括 许多对人类健康很重要。此类控制机制统称为 称为衰减和抗终止。拟议的研究将调查 RNA结合蛋白识别并结合特定的机制 RNA 中的位点，以及这些相互作用如何调节转录衰减。 研究的模型系统是 TRAP 蛋白（trp RNA 结合衰减 Protein），一种RNA结合蛋白，调节转录衰减 枯草芽孢杆菌和相关芽孢杆菌中的色氨酸生物合成基因。在 当存在过量色氨酸时，TRAP 被激活，与一系列 11 GAG 或 UAG 在 trp 操纵子的 5' 前导区重复。这个绑定 诱导转录终止子的形成，从而阻止转录终止子的表达 基因。 TRAP 是一种具有 11 个亚基的蛋白质，形成对称环。 RNA 结合 通过包裹蛋白质环的外周来捕获。 TRAP与其RNA靶标结合的详细机制将是 使用平衡结合研究的组合进行表征，核苷 类似物和快速淬灭停流研究。 TRAP被激活以结合RNA 通过结合 11 个 L-色氨酸分子。晶体学、遗传学和 将使用生化方法来确定其机制 色氨酸结合激活 TRAP。第三个目标是开发更多 详细了解 TRAP 介导的转录机制 衰减。 TRP 前导序列中 TRAP 结合位点的关键元素是 改变，并使用体内研究这些变化对衰减的影响 trpE''-'lacZ 基因融合。这些研究将以信息为指导 从 TRAP/RNA 相互作用的体外研究中获悉。