QUANTITATIVE STUDIES OF THE BIOTIN REGULATORY SYSTEM

对遗传信息表达的调控对 任何器官的正常发育和运作。监管在 转录启动的水平已经被证明依赖于 多种蛋白质和蛋白质之间形成的大分子相互作用 DNA上的目标位置。对…的机械理解 因此，转录调控需要详细的量化 有关单个大分子相互作用的信息 为这一进程做出贡献。生物素调控系统来自 大肠埃希菌是一个复杂的转录调控的例子。 一种生理功能依赖于 蛋白质-小分子、同源和异源蛋白质-蛋白质 以及蛋白质与DNA的相互作用。热力学耦合此外， 个体之间存在相互作用。本研究的目的是 是为了阐明结构、流体动力学和动力学规律 生物素转录调控中的生理功能 系统。主要调控因子协同结合的动力学研究 蛋白质，生物阴阻遏物，到DNA上的目标位置，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 滴定与平衡分析超滤法 测量将被用来检查DNA结合到 转录抑制蛋白组装中的蛋白质二聚化 很复杂。参与其中的蛋白质和DNA的结构元素 转录中形成的多个大分子界面 利用化学DNA足迹技术探索阻遏复合体 和化学蛋白质足迹技术。单点突变体 中断其中一个或多个接口将被隔离并 其特征是量化变化的后果 功能能量学的每个界面的结构完整性 系统。最后，生物素抑制物复合体的结晶 结合小配体和丹的提议是这样的，我们可以 最终得到多重泛函的高分辨结构 这种变构位点特异的DNA结合蛋白的状态。结果： 这些关于生物素调节系统的拟议研究将提供 与理解作用机制相关的信息 任何复杂的转录调控系统。

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.