QUANTITATIVE STUDIES OF THE BIOTIN REGULATORY SYSTEM

生物素调节系统的定量研究

• 批准号: 2022498
• 负责人: DOROTHY BECKETT
• 金额: $ 15.51万
• 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 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022498
• 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.

调节遗传信息的表达对 任何构造的适当开发和功能。 调节 已证明转录启动水平取决于 多种蛋白质和 DNA上的目标位点。 机械理解 因此，转录调节需要详细的量化 有关单个大分子相互作用的信息 为过程做出贡献。 来自 大肠杆菌是复杂的转录调节的一个例子 生理功能取决于结合的系统 蛋白质小分子，同源和异源蛋白质蛋白 和蛋白-DNA相互作用。 热力学耦合，此外， 存在于STHE单个互动之间。 这个研究的目标 要阐明结构性，动力学和动力学规则 控制生物素转录调节的生理功能 系统。 主要监管的合作结合动力学 蛋白质，生物素抑制剂，在DNA上的靶位点，生物素 操作员将使用时间解析的DNase足迹来测量 并停止流量荧光。 直接平衡荧光 滴定和均衡分析超速探测 测量将用于检查DNA结合到与 转录抑制组装中的蛋白质二聚化 复杂的。 蛋白质和DNA的结构元素涉及 转录中形成的多个大分子界面 将使用化学DNA足迹探测抑制复合物 和化学蛋白足迹技术。 单位突变体 破坏一个或多个这些接口将被隔离，并且 o的特征量量化了过度的后果 每个接口的结构完整性 系统。最后，生物素抑制剂复合物的结晶 提议与小配体绑定到Dan，以便我们可以 最终获得多重功能的高分辨率结构 这种变构特异性DNA结合蛋白的状态。 结果 这些对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 任何复杂的转录调节系统。