Quantitative Studies of the Biotin Regulatory System

描述（由申请方提供）：蛋白质-蛋白质相互作用至关重要 调节遗传信息的表达。同源性的强度 蛋白质-蛋白质相互作用决定了转录水平的占有率， DNA上的调节位点。异源蛋白质-蛋白质相互作用的模式 确定DNA靶位点的选择和/或DNA靶位点的占据水平， 绝佳的价钱通过它们对DNA结合的影响， 是控制遗传信息表达的基础。阐明 在以下水平成功进行临床干预所需的详细信息 控制基因表达的蛋白质-蛋白质相互作用需要结合 溶液物理化学研究功能以及高分辨率 结构信息。大肠杆菌生物素调节系统提供 一个很好的模型系统，用于详细研究遗传控制， 调节开关通过蛋白质-蛋白质相互作用。本提案中 实验旨在确定的结构和热力学特征， 描述了辅阻遏物诱导的转录阻遏物的装配。 研究通过异源基因调控基因表达的策略 还概述了蛋白质-蛋白质相互作用。所采用的方法 建议的工作包括定点和随机诱变，停流 荧光动力学，沉降平衡，DNA酶I 足迹法、等温滴定量热法和X射线晶体学。 这些综合研究的结果将产生一般信息， 对理解蛋白质-蛋白质的详细化学性质的重要性 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.