Quantitative Studies of the Biotin Regulatory System

描述(申请人提供)：蛋白质-蛋白质相互作用至关重要 对遗传信息表达的调控。同源的力量 蛋白质-蛋白质相互作用决定转录因子的占位水平 DNA上的监管站点。异源蛋白质-蛋白质相互作用模式 确定DNA目标位置的选择和/或 地点。通过它们对DNA结合的影响这些大分子相互作用 是控制遗传信息表达的基础。澄清： 成功的临床干预所需的细节 控制基因表达的蛋白质-蛋白质相互作用需要结合 函数的溶液物理化学研究以及高分辨率 结构信息。大肠埃希菌生物素调控系统提供 一种用于详细研究基因控制的优秀模式系统 通过蛋白质-蛋白质相互作用的调节开关。在本建议书中 为确定结构和热力学特征而设计的实验 描述了辅阻遏子诱导的转录抑制子的组装。 研究通过异源基因调控基因表达的策略 还概述了蛋白质与蛋白质的相互作用。中使用的方法 建议的工作包括定点突变和随机突变、停流 荧光动力学测量、沉淀平衡、DNaseI 足迹法、等温滴定量热法和x射线结晶学。 这些联合研究的结果将产生一般的信息 了解蛋白质-蛋白质的详细化学结构的意义 基因表达调控所必需的相互作用。

DESCRIPTION (provided by applicant): Protein-protein interactions are critical to regulation of expression of genetic information. The strength of homologous protein-protein interactions dictates the level of occupancy of transcriptional regulatory sites on DNA. Patterns of heterologous protein-protein interactions determine the selection of DNA target site and/or the level of occupancy of a site. Through their influence on DNA binding these macromolecular interactions are fundamental to control of expression of genetic information. Elucidation of the details necessary for successful clinical intervention at the level of protein-protein interactions in control of gene expression requires combined solution physico-chemical studies of function as well as high-resolution structural information. The Escherichia coli biotin regulatory system provides an excellent model system for detailed studies of the control of a genetic regulatory switch via protein-protein interactions. In this proposal experiments designed to determine the structural and thermodynamic features of corepressor-induced assembly of a transcriptional repressor are described. Strategies to examine the control of gene expression via heterologous protein-protein interactions are also outlined. Methods utilized in the proposed work include site-directed and random mutagenesis, stopped-flow fluorescence kinetic measurements, sedimentation equilibrium, DNaseI footprinting, isothermal titration calorimetry and x-ray crystallography. Results of these combined studies will yield information of general significance to understanding the detailed chemistry of the protein-protein interactions essential to regulation of genetic expression.