Structural Studies of the Bacterial Transcription Factor NtrC

描述(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，经常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，分子机制的许多组成部分已经被结构表征，包括关键的酶，RNA聚合酶。S54-聚合酶转录系统提供了化学感知与特定基因转录速率变化的直接耦合，这一过程由所需转录激活蛋白中的ATPase活性介导。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ATPase活性的构象变化。ATPase将来自ATP水解的化学能耦合到s54-聚合酶的构象变化中，从而启动转录。对s54亚基的研究正在为结构变化的本质提供洞察力。结合诱导反应和ATP驱动的构象变化的过程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系统。我们的广泛目标是对转录激活子的功能以及它们如何通过s54聚合酶发挥作用提供一个全面的分子水平的了解。我们将继续关注Aquifex Eolicus蛋白，以开发与生化功能的联系，并了解调控机制。我们将扩展S54的结构研究，为完成除N-末端70个氨基酸以外的所有结构提供数据。我们将研究s54的N末端残基是如何与激活蛋白相互作用的，并研究ATP水解驱动导致转录启动的构象变化的机制。利用单分子操纵实验，我们将研究s54对机械力的响应，类似于激活剂施加的响应。S54-转录激活系统存在于大多数细菌中，并参与调节一些关键基因的转录，这些基因影响毒力和改变宿主的能力。它不存在于真核生物中，因此可能成为未来药物开发的目标。通过拟议的工作了解结构力学将极大地帮助这一努力。激活剂的AAA+结构域类似于许多人类蛋白质中帮助重组蛋白质复合体的结构域，这一过程通常不被很好地理解。更好地了解激活的ATPase应该有助于深入了解其他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.