Effector binding by Bacillus subtilit CodY

描述（由申请人提供）：当面临营养限制时，枯草芽孢杆菌采用几种适应性策略。许多这些适应性机制的表达是在革兰氏阳性菌中高度保守的蛋白质CodY的调控下。CodY通过与其效应物GTP和支链氨基酸（BCAAs）的相互作用来感知营养物质的可用性。在效应物结合后，CodY能够结合并抑制其DNA靶标。目前还不确定CodY中哪些氨基酸是与这些效应物相互作用所必需的，也不清楚效应物结合如何改变CodY，使其与DNA相互作用。本项目的目标是解决这两个基本问题。由于CodY已被证明可以调节几种细菌病原体中毒力因子的表达，因此它是新型抗菌治疗的有吸引力的靶标。然而，这条线的治疗的发展将需要检查有关CodY活动的这些基本问题。本申请的第一个目的是鉴定效应子结合所必需的氨基酸。同源性搜索和X射线晶体学研究已经产生了一些线索，CodY残基可能是重要的GTP和BCAA的结合，分别。将对这些残留物进行诱变处理，并确定其对效应物结合的影响。本申请的第二个目的将研究效应子结合如何增加CodY对DNA的亲和力。部分蛋白水解表明，构象变化发生在CodY结合支链氨基酸后，但没有观察到变化后，CodY结合GTP。近紫外圆二色性，荧光分光光度法和傅里叶变换红外光谱将用于确定GTP是否诱导CodY三级结构的细微变化。第三个目标的实验将评估CodY效应子结合丧失对基因表达的生理影响。这些codY突变将被引入B。枯草杆菌染色体，并通过报告基因融合实验和微阵列分析来测定它们的作用。这项工作的结果将使我们能够了解细菌如何调节基因表达以应对营养饥饿。这种调节的破坏可能会导致某些细菌感染的治疗新手段。

DESCRIPTION (provided by applicant): Bacillus subtilis employs several adaptive strategies when faced with nutrient limitation. Expression of many of these adaptive mechanisms is under regulation by CodY, a highly conserved protein in Gram-positive bacteria. CodY senses nutrient availability by interaction with its effectors, GTP and the branched-chain amino acids (BCAAs). Upon effector binding, CodY is able to bind to and repress its DNA targets. It is uncertain which amino acids in CodY are necessary for interaction with these effectors, and it is unclear how effector binding alters CodY such that it interacts with DNA. The goal of this project is to address both of these underlying questions. As CodY has been shown to regulate expression of virulence factors in several bacterial pathogens, it is an attractive target for novel antimicrobial therapy. However, development of this line of therapy will require examination of these basic issues concerning CodY activity. The first aim of this application will identify amino acids essential for effector binding. Homology searches and x-ray crystallography studies have yielded some clues about which CodY residues may be important for GTP and BCAA binding, respectively. These residues will be mutagenized, and their impact on effector binding will be determined. The second aim of this application will investigate how effector binding increases CodY's affinity for DNA. Partial proteolysis has indicated that a conformational change occurs in CodY upon binding to BCAAs, but no change was observed upon CodY binding to GTP. Near-UV circular dichroism, spectrofluorometry, and Fourier transform infrared spectroscopy will be used to identify whether GTP induces a subtle alteration in the tertiary structure of CodY. Experiments in the third aim will assess the physiological impact of the loss of CodY effector binding on gene expression. These codY mutations will be introduced into the B. subtilis chromosome, and their effects will be assayed by reporter fusion experiments and microarray analysis. The results of this work will allow us to understand how bacteria regulate gene expression in response to nutrient starvation. Disruption of this regulation may result in a novel means of treatment of certain bacterial infections.