Structural Studies of the Bacterial Transcription Factor NtrC

细菌转录因子 NtrC 的结构研究

• 批准号: 8050196
• 负责人: DAVID E WEMMER
• 金额: $ 28.68万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 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 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8050196
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATPase Domain; Affect; Amino Acids; Bacteria; Bacterial RNA; Behavior; Binding; Biochemical; Bioinformatics; Cells; Chemicals; Collaborations; Complex; Coupled; Couples; Coupling; Cysteine; DNA; DNA-Directed RNA Polymerase; Data; Elements; Environment; Enzymes; Eukaryota; Event; Future; Genes; Genetic Transcription; Genome; Goals; Grant; Holoenzymes; Human; Infection; Isotope Labeling; Label; Lead; Learning; Ligand Binding; Ligands; Measures; Mechanics; Mediating; Modeling; Molecular; N-terminal; NMR Spectroscopy; Nature; Organism; Phosphorylation; Polymerase; Process; Production; Proteins; Regulation; Signal Transduction; Structural Models; Structure; System; Testing; Transcription Coactivator; Transcription Initiation; Virulence; Virulence Factors; Work; dimer; drug development; environmental change; in vivo; insight; laser tweezer; new therapeutic target; promoter; protein complex; research study; response; single molecule; transcription factor

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.

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，通常改变特定基因的转录水平以改变蛋白质水平。与真核生物相比，脯氨酸的调节相对简单，并且分子机制的许多组分已经在结构上表征，包括关键酶RNA聚合酶。s54-聚合酶转录系统提供了化学传感与特定基因转录速率变化的直接偶联，这是由所需转录激活蛋白中的ATP酶活性介导的过程。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水解产生的化学能偶联到s54聚合酶中的构象变化中，使转录起始成为可能。对s54亚基的研究提供了对结构变化本质的深入了解。结合诱导的反应和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背景下，在这个系统中产生的见解将有助于理解许多其他的。 我们的主要目标是提供一个全面的分子水平上的理解的功能，转录激活因子，以及他们如何通过s54聚合酶。我们将继续专注于Aquifex aeolicus蛋白与生化功能的联系，并了解调控机制。我们将扩展s54的结构研究，提供数据，以完成所有的结构，但N-末端70个氨基酸。我们将研究S54的N-末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。 s54转录激活子系统存在于大多数细菌中，并参与调节影响毒力和改变宿主能力的一些关键基因的转录。它不存在于真核生物中，因此可能是未来药物开发的目标。通过拟议的工作了解结构力学将大大有助于这样的努力。激活剂的AAA+结构域类似于许多人类蛋白质中的此类结构域，其有助于重组蛋白质复合物，这一过程通常不太清楚。更好地了解激活ATP酶应该提供洞察其他AAA+蛋白质的功能。 公共卫生相关性：细胞不断感知环境，并对环境变化做出反应，以优化生存。一个重要的反应是改变基因转录的水平，以调节细胞中蛋白质的浓度。用于感测信号和响应信号的机制高度变化，以提供不同类型信号所需的适当灵敏度和响应速率。我们提出的实验将研究，在结构水平上，如何通过转录激活因子的传感耦合到增加基因转录的细菌RNA聚合酶的特定形式（与s54亚基），给出了一个快速的反应，并显着改变转录水平。这项工作有重叠的目标，了解参与细胞内和细胞周围的化学信号传感，然后改变基因转录的分子过程，并了解ATP水解的能量是如何通过转录激活因子转化为构象变化，调节聚合酶活性。我们学到的原理将为我们提供对许多其他系统的洞察力。s54系统的转录仅发生在细菌中，用于产生对宿主相互作用重要的毒力因子和蛋白质，我们的研究可能为治疗感染的新治疗靶点提供思路。