REGULATION OF THE TRYTOPHAN GENES IN BACILLUS

在编码区之前控制转录终止是一种 常用的策略来调节细菌中的基因表达，包括 许多对人类健康很重要。这些控制机制共同 称为减毒和抗终止。拟议的研究将调查 RNA结合蛋白识别和结合特异性 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 蛋白），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色氨酸生物合成基因。在 过量色氨酸的存在下，TRAP被激活以结合一系列11个 在trp操纵子的5'前导区中的GAG或UAG重复。此绑定 诱导转录终止子的形成，其停止转录终止子的表达。 基因. TRAP是一种11个亚基的蛋白质，形成一个对称的环。RNA结合于 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在下文中介绍。 使用平衡结合研究、核苷 类似物和快速骤冷停流研究。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 生物化学方法将用于确定 色氨酸结合激活TRAP。第三个目标是发展一个更 详细了解TRAP介导的转录机制 衰减trp前导序列中TRAP结合位点的关键元件将是 改变，这些变化对衰减的影响在体内研究，使用 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了解到。

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.