QUANTITATIVE STUDIES OF THE BIOTIN REGULATORY SYSTEM

生物素调节系统的定量研究

• 批准号: 6028316
• 负责人: DOROTHY BECKETT
• 金额: $ 5.17万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6028316
• 关键词: DNA; DNA footprinting; adenosine monophosphate; allosteric site; biotin; chemical binding; crystallization; genetic operator element; intermolecular interaction; nucleic acid sequence; time resolved data

Regulation of the expression of genetic information is critical to the proper development and functioning of any organsim. Regulation at the level of transcription initiation has been demonstrated to depend on macromolecular interactions formed between multiple proteins and target sites on the DNA. A mechanistic understanding of transcriptional regulation therefore requires detailed quantiative information about the individual macromolecular interactions that contribute to the process. The biotin regulatory system from Escherichia coli is an example of a complex transcriptional regulatory system in which physiological function depends on a combination of protein-small molecule, homologous and heterologous protein-protein and protein-DNA interactions. Thermodynamic coupling moreover, exists between sthe individual interactions. The goals of this reserch are to elucidate the structural, thedrmodynamic and kinetic rules that govern the physiological function in the biotin transcriptional regulatory system. The kinetics of cooperative binding of the major regulatory protein, the bioyin repressor, to its target site on the DNA, the biotin operator, will be measured using Time Resolved Dnase Footprinting and Stopped-Flow Fluorescence. Direct Equilbrium Fluorescence Titrations and Equilibrium Analytical Ultracentrigugation Measurements will be utilized to examine coupling of DNA binding to protein dimerization in assembly of the transcriptional repression complex. The structural elements of the protein and DNA involved in the multiple macromolecular interfaces formed in the transcriptional repression complex will be probed using Chemical DNA Footprinting and Chemical Protein Footprinting techniques. Single site mutants that disrupt one or more of these interfaces will be isolated and characterized o quantitate the consequences of altrations in the structural integrity of each interface for the functional energetics of the system. Finally, crystallization of complexes of the biotin repressor bound to small ligands and to DAN is proposed so that we may ultimately obtain high resolution structures of the multiple functional states of this allosteric site-specific DNA binding protein. Results of these proposed studies of the biotin regulatory system will provide information that is relevant to understanding the mechanism of function of any complex transcriptional regulatory system.

遗传信息表达的调控对于 任何有机体的正常发育和运作。 监管于 转录起始水平已被证明取决于 多种蛋白质之间形成的大分子相互作用 DNA 上的目标位点。 机械性的理解 因此转录调控需要详细的定量 有关单个大分子相互作用的信息 为这一过程做出贡献。 生物素调节系统来自 大肠杆菌是复杂转录调控的一个例子 系统中的生理功能取决于以下因素的组合 蛋白质-小分子、同源和异源蛋白质-蛋白质 以及蛋白质-DNA 相互作用。 此外，热力学耦合， 存在于个体之间的相互作用。 本研究的目标 旨在阐明结构、热力学和动力学规则 控制生物素转录调节的生理功能 系统。 主要监管机构合作约束的动力学 蛋白质（生物素阻遏物）到达 DNA 上的目标位点（生物素） 操作员，将使用时间分辨 DNA 足迹进行测量 和停流荧光。 直接平衡荧光 滴定和平衡分析超速离心 测量将用于检查 DNA 结合的耦合 转录抑制组装中的蛋白质二聚化 复杂的。 参与蛋白质和DNA的结构元件 转录过程中形成的多个大分子界面 将使用化学 DNA 足迹来探测抑制复合物 和化学蛋白质足迹技术。 单位点突变体 破坏这些接口中的一个或多个将被隔离并且 特征化 o 量化改变的后果 每个界面的结构完整性，以实现功能能量学 系统。最后，生物素阻遏物复合物的结晶 建议与小配体和 DAN 结合，以便我们可以 最终获得多功能的高分辨率结构 这种变构位点特异性 DNA 结合蛋白的状态。 结果 这些生物素调节系统的拟议研究将提供 与理解功能机制相关的信息 任何复杂的转录调控系统。