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应对环境变化，通常会改变从特定基因的转录水平以改变蛋白质水平。与真核生物相比，核调节相对简单，并且在结构上表征了分子机械的许多组件，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耦合，这是由ATPase活性在所需的转录激活蛋白中介导的过程。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ATPase活性的构象变化。 ATPase将ATP水解的化学能与S54-聚合酶中的构象变化相结合，从而实现转录启动。 S54亚基的研究正在提供有关结构变化本质的见解。结合引起的反应的过程以及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，该系统中产生的见解也将有助于了解许多其他情况。 我们的广泛目标是对转录激活剂功能以及它们如何通过S54聚合酶作用提供全面的分子水平理解。我们将继续专注于Aquifex Aeolicus蛋白，以与生化功能建立联系，并了解调节机制。我们将扩展S54的结构研究，提供数据以完成除N末端70氨基酸以外的所有结构。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水解驱动导致转录启动的构象变化的机制。使用单分子操纵实验，我们将研究S54对机械力的响应，类似于激活剂所应用的力。 S54转录激活因子系统发生在大多数细菌中，并参与调节某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，因此可能是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样做。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，这些蛋白有助于重新组织蛋白质复合物，通常不太了解的过程。更好地了解激活剂ATPase应提供有关其他AAA+蛋白质功能的见解。 公共卫生相关性：细胞不断感知其环境并响应其变化以优化生存。一个重要的反应是改变基因转录水平以调节细胞中蛋白质的浓度。传感信号和对它们的响应的机制高度变化，以提供不同类型信号所需的适当敏感性和响应速率。我们提出的实验将在结构水平上研究转录激活剂通过细菌RNA聚合酶（带有S54亚基）的特异性提高基因转录的传感，从而产生快速响应并大大改变转录水平。这项工作具有重叠的目标，即了解与细胞内和周围感测化学信号的分子过程，然后改变基因转录，并​​了解转录激活剂如何将ATP水解的能量转化为调节聚合酶活性的构象变化。我们学到的原则将为许多其他系统提供洞察力。仅在细菌中发生的S54系统的转录用于生产毒力因子和蛋白质对宿主相互作用很重要的蛋白质，我们的研究可能为治疗感染的新治疗靶点提供想法。