STRUCTURAL STUDIES OF THE BACTERIAL TRANSCRIPTION FACTOR

细菌转录因子的结构研究

• 批准号: 7030185
• 负责人: DAVID E WEMMER
• 金额: $ 7.94万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7030185
• 关键词: DNA; beryllium; binding sites; biological signal transduction; computer simulation; conformation; crystallization; electron spin resonance spectroscopy; fluoride ion; intermolecular interaction; molecular dynamics; molecular site; nitrogen metabolism; nuclear magnetic resonance spectroscopy; phosphorylation; prokaryote; protein protein interaction; protein structure function; structural biology; transcription factor

Two component signaling systems are very common in bacteria, but also occur in eukaryotes. The signaling occurs through transfer of a phosphate from a kinase to a "receiver" protein, initiating a conformational change, leading to altered interactions with other proteins to propagate the signal. The investigators will study the bacterial transcription factor NtrC, which upon phosphorylation undergoes self-association, interaction with sigma54 polymerase and ATP hydrolysis, leading to transcription. Understanding this relatively simple system will provide a basis for understanding more complex eukaryotic signaling processes, and could also lead to approaches for interrupting specific bacterial signaling processes. The goal of the work proposed here is to determine the structures of the bacterial transcription factor NtrC, and domains of it, to gain an understanding of the molecular basis for its activity, and thereby the general processes for molecular signal transduction. Specific aims are to: (1) determine a high resolution structure of the BeF3- activated receiver domain, and to understand differences and similarities in changes upon activation with respect to other receiver domains; (2) to express and purify the wild type sequence DNA binding domain, and determine the structure of this dimer bound to an enhancer DNA sequence, as well as trying to understand the role of the linker connecting to the adjacent domain; (3) to make and screen constructs of the central ATPase domain for structure determination (including those from a thermophile), and determine the structures of this domain in different nucleotide ligation states; (4) to examine the role of the communication helix linking the receiver and central domains, and to analyze broadly the interactions between the receiver and central domains; (5) to examine intact NtrC with gel shift, fluorescence and EPR approaches to track changes upon activation, assembly and nucleotide hydrolysis, and to express the sigma54 subunit of polymerase to start to explore its interactions with NtrC.

两种成分的信号系统在细菌中非常常见，但是 也存在于真核生物中。信号传导通过磷酸盐的转移发生 从激酶到“接收者”蛋白质，启动构象变化， 导致与其他蛋白质的相互作用改变以传播信号。 研究人员将研究细菌转录因子 NtrC，它 磷酸化后发生自缔合，与 sigma54 相互作用 聚合酶和 ATP 水解，导致转录。了解这一点 相对简单的系统将为理解更复杂的系统提供基础 真核信号传导过程，也可能导致寻找方法 中断特定的细菌信号传导过程。工作目标 这里提出的是确定细菌转录的结构 NtrC 因子及其域，以了解分子基础 其活性，以及​​分子信号的一般过程 转导。具体目标是：（1）确定高分辨率结构 BeF3 激活的接收域，并了解差异和 与其他接收者域激活时变化的相似性； (2)表达并纯化野生型序列DNA结合结构域，以及 确定与增强子 DNA 序列结合的二聚体的结构，如 以及尝试了解连接到相邻的连接子的作用 领域; (3) 制备并筛选中央 ATPase 结构域的构建体 结构测定（包括来自嗜热菌的结构），并确定 该结构域在不同核苷酸连接状态下的结构； (4) 至 检查连接接收器和中央的通信螺旋的作用 领域，并广泛分析接收者和 中心域； (5) 通过凝胶位移、荧光和检测完整的 NtrC EPR 方法跟踪激活、组装和核苷酸变化 水解，并表达聚合酶的 sigma54 亚基，开始聚合 探索其与 NtrC 的相互作用。