Structural Studies of the Bacterial Transcription Factor NtrC

描述（由申请人提供）：调节基因转录的过程是控制所有类型细胞行为的基础。在真核生物中，调控通过非常大的复合物的合作形成而发生，这使得过程的详细表征非常困难。在原核生物中，调控更简单，并且执行这些过程的分子机器的许多组件已经在结构上被表征，包括RNA聚合酶及其与a70的复合物。我们对转录激活因子和a54的研究扩展了对细菌转录调控的理解。在细菌中，对环境变化的感知和响应通常通过双组分系统发生，激酶-受体结构域对，其中大多数导致转录水平的改变。这些系统相对简单，允许研究涉及转录控制和启动的基本过程。我们的长期目标是提供一个全面的分子水平的理解，一个双组分系统，通过54聚合酶的行为。我们将继续对转录激活因子NtrC、NtrC 1和NtrC 4进行结构研究，以了解磷酸化驱动的构象变化如何导致活性寡聚体的组装以及随后与α 54的相互作用，ATP水解如何改变NtrC的构象，从而改变α 54-聚合酶的构象以使DMA打开，这些蛋白质的序列特异性DNA结合的基础以及DNA结合中的协同性如何发生。我们还将确定a54结构域的结构，确定它们如何相互作用，并允许我们对其他人在低分辨率下确定的NtrC-a54复合物进行建模。我们还将分析一个同系物NtrC 2，它包含一个GAP结构域，而不是一个接收器结构域来调节它，看看调节机制是否与NtrC 1相同。我们将使用NMR光谱来确定结构和探测动力学域，和X射线晶体学，以确定所需的较大的复合物的结构。双组分系统发生在原核生物和低等真核生物中，因此，为未来的药物开发提供了可能的靶点，通过这项工作获得的结构理解将有所帮助。正在研究的蛋白质的其他部分与帮助重组蛋白质复合物的人类蛋白质非常相似。这些工作的细节尚不清楚，从这些细菌版本的结构研究中学到的东西将提供对这些功能的见解。基因调控对生命过程至关重要，因此必须详细了解它，以便能够通过疾病或环境的影响了解过程中改变的影响。

DESCRIPTION (provided by applicant): Processes that regulate gene transcription are fundamental in controlling the behavior of cells of all types. In eukaryotes regulation occurs through cooperative formation of very large complexes, which make detailed characterization of the processes very difficult. In prokaryotes regulation is simpler, and many components of the molecular machinery that execute the processes have been structurally characterized, including the RNA polymerase and its complex with a70. Our studies of transcriptional activators and a54 expand the understanding of bacterial transcriptional regulation. In bacteria sensing and responding to environmental changes often occurs through two-component systems, kinase - receiver domain pairs, most of which lead to altered levels of transcription. These systems are relatively simple, allowing study of the basic the processes involved in control and initiation of transcription. Our long term goal is to provide a comprehensive molecular level understanding of a two- component system that acts through a54 polymerase. We will continue structural work on the transcriptional activators NtrC, NtrC1 and NtrC4, to understand how the phosphorylation driven conformational change leads to assembly of the active oligomer and subsequent interaction with a54, how ATP hydrolysis changes the conformation of the NtrCs and, thereby that of the a54-polymerase to enable opening of the DMA, the basis for sequence specific DMA binding by these proteins and how cooperativity in DMA binding occurs. We will also determine structures of a54 domains determine how they interact, and to allow us to model NtrC-a54 complexes that are being determined by others at low resolution. We will also analyze a homolog NtrC2, which contains a GAP domain instead of a receiver domain to regulate it, to see if the regulatory mechanism is the same as in NtrC1. We will use NMR spectroscopy to determine structures and probe dynamics of domains, and x-ray crystallography to determine structures of larger complexes as needed. Two component systems occur in prokaryotes and lower eukaryotes and, hence, provide possible targets for future drug development, which the structural understanding obtained through this work would aid. Other parts of the proteins being studied are very similar to human proteins that help reorganize protein complexes. The details of how these work are not yet clear, and what is learned from structural studies of these bacterial versions will provide insights into how these function. Gene regulation is so central to the processes of life that it is important to understand it in great detail, to be able to understand the implications of alterations in the process through effects of disease or the environment.