PHYSICAL AND GENETIC STUDIES OF REPRESSOR PROTEINS

这项研究项目旨在调查 蛋白质识别DNA的特定序列，以及小分子 配体调节识别过程。乳胶和色氨酸抑制物来自 E.Coli代表可诱导和可诱导的调节蛋白 阻遏系统；这两种蛋白质及其靶DNA序列 可以分离出所需的物理量和化学量 研究，编码区在特定部位的载体上可用 突变和其他基因操作。具体目标是： (1)评估特定的氨基酸和区域的作用 这些阻遏蛋白功能的初级序列。这 检查将包括根据基因序列产生定点突变体 色氨酸抑制物X-射线晶体结构和已知化学物质， 乳胶抑制物的物理和同源性数据。交汇处 与DNA结合有关的螺旋-转角-螺旋结构 抑制者将被采取。这些特别设计的蛋白质和 可从其他来源获得的突变蛋白将在 细节。针对这些主要序列的特定片段的抗体 会产生蛋白质，而抗体-抑制物复合体的作用 将对功能性活动的形成进行测量。(2)确定 DNA结构对结合活性的影响。的影响 抑制子-DNA(操纵子和)结合参数的超螺旋 非特异性结合)将被检查。蛋白质与DNA的交联 将尝试评估蛋白质中的特定区域 非常接近DNA。(3)蛋白质结构的热力学研究 功能。阻遏蛋白的高压解离及其影响 在这个过程中的配基将提供有关强度的信息 亚基间接触及配体在这些接触中的变化 有约束力的。蛋白质变性的量热测量(DSC)和 将进行配体结合以评估其热力学性质 蛋白质及其相互作用。(4)蛋白质的测定 结构和构象变化。分子的荧光光谱 将测量本征和外在荧光团，以及核磁共振光谱 将被用来检测色氨酸抑制物的结合性质。 从这些研究中获得的数据在两个不同的 在文献中提供数据的系统将扩展我们对 基因调控是所有生物体的基本功能。

This research project is designed to investigate the mechanisms by which proteins recognize specific sequences of DNA and the means by which small ligands modulate the recognition process. The lac and trp repressors from E. coli represent regulatory proteins in the classes of inducible and repressible systems; both of these proteins and their target DNA sequences can be isolated in the quantities required for physical and chemical studies, and the coding regions are available on vectors for site-specific mutagenesis and other genetic manipulations. The specific objectives are: (1) assessment of the roles of specific amino acids and regions of the primary sequences in the function of these repressor proteins. This examination will include generation of site-specific mutants based on the trp repressor x-ray crystallographic structure and on the known chemical, physical and homology data for lac repressor. Interchange of the helix-turn-helix structure implicated in DNA binding between the two repressors will be undertaken. These specifically designed proteins and mutant proteins available from other sources will be characterized in detail. Antibodies to specific segments of the primary sequence of these proteins will be generated, and the effects of antibody-repressor complex formation on functional activities will be measured. (2) Determination of the effects of DNA structure on binding activities. The effects of supercoiling on the binding parameters for repressor-DNA (both operator and nonspecific binding) will be examined. Cross-linking of proteins to DNA will be attempted to assess specific regions of the protein which are in close proximity to DNA. (3) Thermodynamic studies of protein structure and function. High pressure dissociation of the repressor proteins and effects of ligands on this process will provide information about the strength of inter-subunit contacts and alterations in these contacts by ligand binding. Calorimetric measurements of protein denaturation (DSC) and ligand binding will be undertaken to assess the thermodynamic properties of the proteins and their interactions. (4) Determination of protein structure and conformational changes. FLuorescence spectroscopy of intrinsic and extrinsic fluorophores will be measured, and NMR spectroscopy will be utilized to examine the binding properties of the trp repressor. The correlation of data obtained from these studies on two different systems with data available in the literature will expand our knowledge of genetic regulation, an essential function in all living organisms.