STRUCTURAL STUDIES OF THE BACTERIAL TRANSCRIPTION FACTOR

两种信号系统在细菌中很常见，但是 也存在于真核生物中。该信号通过磷酸盐的转移发生 从一种激酶变成一种“接受者”蛋白质，启动构象变化， 导致与其他蛋白质的相互作用发生变化，从而传播信号。 研究人员将研究细菌转录因子NTRC，它 一旦磷酸化，就会自我结合，与Sigma54相互作用 聚合酶和三磷酸腺苷的水解，导致转录。理解这一点 相对简单的系统将为理解更复杂的系统提供基础 真核信号传递过程，也可能导致方法 干扰特定的细菌信号传递过程。这项工作的目标 这里提出的是确定细菌转录的结构 因子NTRC及其结构域，以获得对分子基础的理解 因为它的活性，从而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激活的接收器结构域，并了解差异和 激活时相对于其他接收域的变化的相似性； (2)表达和纯化野生型序列DNA结合域，以及 确定与增强子DNA序列结合的二聚体的结构，如下 以及试图理解链接器连接到相邻 结构域；(3)制造和筛选中央ATPase结构域以用于 结构测定(包括来自嗜热菌的那些)，并确定 该结构域在不同核苷酸连接状态下的结构； 检查连接接收器和中心的通信螺旋的作用 域，并广泛地分析接收者和 中心结构域；(5)用凝胶位移、荧光和荧光检测完整的NTRC EPR方法跟踪激活、组装和核苷酸的变化 水解酶，并表达聚合酶的sigma54亚单位开始 探索其与NTRC的互动。

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.