QUANTITATIVE STUDIES OF THE BIOTIN REGULATORY SYSTEM

生物素调节系统的定量研究

• 批准号: 2701549
• 负责人: DOROTHY BECKETT
• 金额: $ 10.9万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE COUNTY
• 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 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2701549
• 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人类的免疫系统至关重要。 任何器官的正常发育和功能。 调控 转录起始的水平已被证明依赖于 多个蛋白质之间形成的大分子相互作用， DNA上的靶点 机械地理解 因此，转录调控需要详细的定量分析。 关于单个大分子相互作用的信息， 为进程做出贡献。 生物素调节系统来自 大肠杆菌是一个复杂的转录调控的例子， 生理功能取决于以下因素组合的系统 蛋白质-小分子、同源和异源蛋白质-蛋白质 和蛋白质-DNA相互作用。 此外，热力学耦合， 存在于个体之间的相互作用。 本研究的目的是 是为了阐明结构，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 主要调节因子的协同结合动力学 蛋白质，生物素阻遏物，到其在DNA上的靶位点，生物素 将使用时间分辨DNA酶足迹法测量 和停流荧光。 直接平衡荧光 滴定和平衡分析超离心 将利用测量来检查DNA结合与DNA结合的偶联。 蛋白质二聚体在转录抑制组装中的作用 复杂. 蛋白质和DNA的结构元件参与了 转录过程中形成的多个大分子界面 将使用化学DNA足迹法探测阻遏复合物 化学蛋白质足迹技术 单位点突变体， 中断这些接口中的一个或多个将被隔离， 其特征在于量化的变化的后果， 每个界面的功能能量学的结构完整性 系统最后，生物素阻遏物复合物的结晶 结合到小配体和DAN的建议，使我们可以 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。