PHYSICAL AND GENETIC STUDIES OF REPRESSOR PROTEINS

本研究项目旨在研究 蛋白质识别DNA的特定序列， 配体调节识别过程。 lac和trp阻遏物 e.大肠杆菌代表了可诱导和 阻遏系统;这些蛋白质及其靶DNA序列 可以分离出物理和化学所需的量， 研究，并且编码区可在载体上用于位点特异性 诱变和其他遗传操作。 具体目标是： (1)评估特定氨基酸和区域的作用， 这些阻遏蛋白功能的主要序列。 这 检查将包括基于以下的位点特异性突变体的产生： Trp阻遏物的X射线晶体结构和已知的化学物质， Lac阻遏物的物理和同源性数据。 立交 螺旋-转角-螺旋结构与DNA结合有关 将采取镇压措施。 这些专门设计的蛋白质和 可从其它来源获得的突变蛋白的特征在于 详细 针对这些抗体的一级序列的特定片段的抗体， 蛋白质将产生，抗体-阻遏物复合物的作用 将对职能活动的培训进行衡量。 (2)测定 DNA结构对结合活性的影响。 的影响 超螺旋对阻遏物-DNA的结合参数（操纵子和 非特异性结合）。 蛋白质与DNA的交联 将试图评估蛋白质的特定区域， 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 配体在这一过程中将提供有关强度的信息 亚基间接触和配体对这些接触的改变 约束力 蛋白质变性的量热测量（DSC）和 配体结合将进行评估的热力学性质 蛋白质及其相互作用。 (4)测定蛋白质 结构和构象变化。 荧光光谱 将测量内在和外在的荧光团，并且NMR光谱 将被用于检查Trp阻遏物的结合特性。 从这些研究中获得的两个不同的数据的相关性 系统与文献中的数据将扩大我们的知识 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.