Structural Studies of the Bacterial Transcription Factor NtrC

描述（由申请人提供）：细菌使用许多不同的蛋白质来感知和响应环境变化，通常通过改变特定基因的转录水平来改变蛋白质水平。与真核生物相比，原核生物的调控相对简单，许多分子机制的组成部分已经在结构上得到了表征，包括关键酶RNA聚合酶。554聚合酶转录系统提供了化学感应与特定基因转录速率变化的直接耦合，这一过程是由所需转录激活蛋白中的atp酶活性介导的。我们对这些激活蛋白的研究表明，接收信号（磷酸化或配体结合）如何导致激活atp酶活性的构象变化。ATP酶将ATP水解产生的化学能耦合到554聚合酶的构象变化中，从而使转录启动成为可能。对s54亚基的研究为结构变化的本质提供了洞见。结合诱导反应的过程和ATP驱动的构象变化发生在所有生物体和许多不同的环境中，在这个系统中产生的见解也将有助于理解许多其他的。我们的广泛目标是提供一个全面的分子水平的转录激活剂的功能和他们如何通过s54聚合酶的作用的理解。我们将继续关注水蛭蛋白，以开发与生化功能的联系，并了解调节机制。我们将扩展s54的结构研究，提供数据来完成除n端70个氨基酸外的所有氨基酸的结构。我们将研究s54的n端残基如何与激活蛋白相互作用，并研究ATP水解驱动导致转录起始的构象变化的机制。使用单分子操作实验，我们将研究s54对机械力的响应，类似于激活剂施加的机械力。554转录激活器系统存在于大多数细菌中，并参与调节一些影响毒力和改变宿主能力的关键基因的转录。它不会发生在真核生物中，因此可能成为未来药物开发的目标。通过提出的工作了解结构力学将极大地帮助这一努力。激活剂的AAA+结构域与许多人类蛋白质中的AAA+结构域相似，这些结构域有助于重组蛋白质复合物，这一过程通常不被很好地理解。对激活物atp酶的更好理解将有助于进一步了解其他AAA+蛋白的功能。

DESCRIPTION (provided by applicant): Bacteria use many different proteins to sense and respond to environmental changes, often altering levels of transcription from specific genes to alter protein levels. Prokaryotic regulation is relatively simple compared to eukaryotic, and many components of the molecular machinery have been structurally characterized, including the key enzyme, RNA polymerase. The s54-polymerase transcription system provides a direct coupling of chemical sensing to changes in rates of transcription at specific genes, a process mediated by an ATPase activity in required transcriptional activator proteins. Our studies of these activator proteins have shown how receiving a signal (phosphorylation or ligand binding) leads to conformational changes that activate ATPase activity. The ATPase couples chemical energy from ATP hydrolysis into conformational changes in s54-polymerase that enable transcription initiation. Studies of the s54 subunit are providing insights into the nature of the structural changes. The processes of binding-induced response, and ATP driven conformational changes occur in all organisms and many different contexts, the insights generated in this system will help understand many others as well. Our broad goal is to provide a comprehensive molecular level understanding of the function of transcriptional activators and how they act through s54 polymerase. We will continue to focus on Aquifex aeolicus proteins to develop connections with biochemical function, and to understand regulatory mechanisms. We will extend structural studies of s54, providing data to complete a structure of all but the N-terminal 70 amino acids. We will examine how the N-terminal residues of s54 interact with activator proteins, and study the mechanism by which ATP hydrolysis drives the conformational changes that lead to transcription initiation. Using single molecule manipulation experiments we will investigate the response of s54 to mechanical forces, analogous to that applied by the activators. The s54-transcriptional activator system occurs in most bacteria, and is involved in regulating transcription of some key genes that affect virulence and the ability to change hosts. It does not occur in eukaryotes, and hence could be a target for future drug development. Understanding structural mechanics through the proposed work would greatly aid such an effort. The AAA+ domain of the activators is similar to such domains in many human proteins that help reorganize protein complexes, processes that are generally not well understood. Better understanding of the activator ATPase should provide insights into function of other AAA+ proteins. PUBLIC HEALTH RELEVANCE: Cells constantly sense their environment and respond to changes in it to optimize survival. One important response is altering the level of gene transcription to modulate the concentrations of proteins in the cell. The mechanisms for both sensing signals and responding to them are highly varied to provide the appropriate sensitivity and rate of response required for different types of signals. The experiments we propose will study, at the structural level, how sensing by transcriptional activators is coupled to increasing gene transcription by a specific from of bacterial RNA polymerase (with the s54 subunit) that gives a rapid response and dramatically changes the level of transcription. This work has the overlapping goals of understanding the molecular processes that are involved in sensing chemical signals in and around cells and then altering gene transcription, and understanding how the energy of ATP hydrolysis is converted by the transcriptional activators into conformational changes that modulate polymerase activity. The principles that we learn will provide insight into many other systems. Transcription by the s54 system, which occurs only in bacteria, is used for production of virulence factors and proteins important for host interactions, and our studies may provide ideas for new therapeutic targets to treat infections.