QUANTITATIVE STUDIES OF THE BIOTIN REGULATORY SYSTEM

原核生物和真核生物中基因表达的调控 受复杂的大分子组装的影响。这些产品的功能 组装可以依赖于蛋白质-蛋白质，蛋白质-小分子， 以及蛋白质与DNA的相互作用。生物素生物合成的调控 而在大肠杆菌中的滞留取决于这三种类型的 互动。生物素系统的模型，基于一个 对大分子过程的定性理解 促进监管，已被提出。生物素系统 为物理化学的关联提供了一个理想的模型系统 单个大分子相互作用对生物的影响 复杂的基因调控系统。这项研究的目标是 建议的目标是： 1.对影响相互作用的因素进行量化研究 生物素调节来定义细胞的功能能量学 生物素系统。 2.调查造成这种现象的热力学驱动力 监管互动。 3.对生物素调控的定性模型进行评估和修正。 根据物理研究和开发的结果， 测试系统的量化模型。 将在这些研究中使用的技术包括。 测量DNA结合的定量DNA酶足迹技术， B.平衡沉淀法测定亚基组装 性质，以及C.用于测量的荧光光谱 异源蛋白质-蛋白质相互作用。这些研究都是关于 生物素系统将有助于定义 个体生物素调节相互作用的物理化学和 整个系统的生物学，并可能揭示一般原理 这与理解更复杂的基因调控有关 系统。相互作用的热力学表征将会， 此外，产生有关物理化学力的信息 推动这些互动。

Regulation of gene expression in prokaryotes and eukaryotes is effected by complex macromolecular assemblies. Function of these assemblies can depend on protein-protein, protein-small molecule, and protein-DNA interactions. Regulation of biotin biosynthesis and retention in E. coli depends on all three types of interactions. A model for the biotin system, based on a qualitative understanding of the macromolecular processes that contribute to the regulation, has been proposed. The biotin system provides an ideal model system for relating the physical chemistry of individual macromolecular interactions to the biology of a complex gene regulatory system. The goals of this research proposal are to: 1. perform quantitative studies of the interactions that contribute to biotin regulation to define the functional energetics of the biotin system. 2. investigate the thermodynamic driving forces responsible for the regulatory interactions. 3. evaluate and revise the qualitative model for biotin regulation in light of the results of the physical studies and develop and test quantitative models for the system. The techniques that will be employed in these studies include a. the quantitative DNAse footprint technique to measure DNA binding, b. equilibrium sedimentation to determine of subunit assembly properties, and c. fluorescence spectroscopy to measure heterologous protein-protein interactions. These studies of the biotin system will help define the relationship between the physical chemistry of individual biotin regulatory interactions and the biology of the entire system and may reveal general principles that are relevant to understanding more complex gene regulatory systems. Thermodynamic characterization of the interactions will, moreover, yield information about the physical chemical forces that drive these interactions.