ABRB BINDING TO DEVELOPMENTALLY CONTROLLED PROMOTERS

ABRB 与发育控制启动子的结合

• 批准号: 6018868
• 负责人: MARK A STRAUCH
• 金额: $ 22.49万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6018868
• 关键词: Bacillus subtilis; DNA binding protein; DNA directed RNA polymerase; DNA footprinting; X ray crystallography; bacterial genetics; bacterial proteins; chemical fingerprinting; conformation; developmental genetics; gene mutation; genetic promoter element; microorganism culture; nuclear magnetic resonance spectroscopy; protein structure function; site directed mutagenesis

The principal investigator proposes to study the AbrB protein Bacillus subtilis. AbrB is a key global regulator which adjusts gene expression to fit metabolic needs in suboptimal environments, stress and the initial stages of the developmental process of sporulation. In addition to preventing inappropriate expression of stationary phase associated functions during rapid growth, recent evidence suggests that AbrB also plays a role in modulating catabolite repression and could affect growth-rate dependent regulation of components of the translational apparatus. AbrB is a transcriptional regulator whose N-terminal domain represents a novel DNA-binding motif that primarily recognizes subtle three-dimensional DNA structures that are assumed by a subset of varying sequences. The PI argues that elucidation of the factors responsible for flexible AbrB binding specificity will provide insights in protein-DNA recognition mechanisms and how a cell can economically use a single protein to coordinate a variety of stress responses and developmental options. Currently, over 35 genes encoding a wide array of metabolic functions, including some essential for normal sporulation, are known to have AbrB binding sites, usually in the promoter regions. To better define the exact structural parameters of the DNA targets recognized by AbrB, binding to select sites will be probed by high resolution footprinting techniques and kinetics examined using a sensitive real-time assay. The relationship of protein structure to DNA- binding properties will be probed by a combination o mutant analysis, NMR of isolated DNA-binding domain, C domain multimerization studies and crystallography of AbrB-DNA complexes. In vitro transcription and binding assays will test hypotheses regarding AbrB-mediated regulatory mechanisms at representative promoters where different classes of AbrB-dependent effects are suspected. Where necessary, confirmatory in vivo genetic evidence will be obtained.

主要研究者建议研究AbrB蛋白芽孢杆菌 枯草芽孢杆菌AbrB是调节基因表达的关键全局调节因子 以适应次优环境中的代谢需求，压力和最初的 孢子形成过程的各个阶段。除了 防止不适当的表达稳定相相关 在快速生长过程中发挥作用，最近的证据表明， 也在调节分解代谢物抑制中起作用， 生长速率依赖性调节的组件的翻译 设备. AbrB是一种转录调节因子，其N端结构域 代表了一种新的DNA结合基序， 三维DNA结构，由一个子集假设， 不同的序列。PI认为，阐明这些因素 负责灵活的AbrB结合特异性将提供见解， 蛋白质-DNA识别机制以及细胞如何经济地 使用单一蛋白质来协调各种应激反应， 发展选择。目前，超过35个基因编码广泛的阵列 包括一些对正常孢子形成至关重要的代谢功能， 已知具有AbrB结合位点，通常在启动子区。 为了更好地确定DNA靶点的确切结构参数， 被AbrB识别，与选择位点的结合将被高 分辨率足迹技术和动力学检查使用 灵敏的实时测定。蛋白质结构与DNA的关系- 结合特性将通过突变体分析的组合来探测， 分离的DNA结合结构域的NMR，C结构域多聚化研究 和AbrB-DNA复合物的晶体学。体外转录和 结合试验将检验关于AbrB介导的 代表性发起人的监管机制， 怀疑存在AbrB依赖性效应。在必要的情况下， 将获得确认的体内遗传证据。