SITE SPECIFIC ENERGETICS OF PROTEIN/DNA INTERACTIONS

这项研究计划的目标是阐明 蛋白质相互作用介导的基因调控过程 DNA上的特定位置。该方案的组成部分包括：(A)发展 研究特定部位的蛋白质-DNA相互作用的新技术， (B)特定体系中的功能能量学的实验研究，(C) 分子的能量性质与其结构特征的关系 相互作用的分子，以及(D)开发和测试物理- 基因控制的化学模型。 我们计划扩展我们之前关于抑制-操纵者控制的研究 噬菌体Lambda系统，这是一个大家族的原型 协作式多点基因控制系统。使用以下技术 量热法结合定量DNA酶足迹滴定和 我们开发的一种新的低温凝胶移动技术，我们将携带 对温度、酸碱度和盐分的影响进行了详细研究 特定部位能量学上的相互作用(包括协作性 Ci和cro抑制物结合到DNA操纵子位点的作用)。这个 这些结果将为质子的详细作用提供新的见解 结合，盐结合和各种非共价力负责 监管方面的相互作用。这些研究将扩展到一系列 缺乏协作性的突变抑制子。 我们将研究合作互动的生物学作用 突变型操纵子结合抑制子的体内外研究 利用一种物理化学手段改变协作性的抑制物 在这个计划下正在开发的基因控制模型 研究。

The goal of this research program is to elucidate physical mechanisms of gene control processes mediated by the interactions of proteins at specific sites on DNA. Components of the program include: (a) development of new techniques for the study of site-specific protein-DNA interactions, (b) experimental studies of functional energetics in specific systems, (c) correlation of the energetic properties with structural features of the interacting molecules, and (d) development and testing of physical- chemical models for gene control. We plan to extend our previous studies on the repressor-operator control system of bacteriophage lambda, which is a prototype for a large family of cooperative multi-site gene control systems. Using techniques of calorimetry in combination with quantitative DNase footprint titration and a new cryogenic gel shift technique that we have developed, we will carry out a detailed study of the effects of temperature, pH, and salt interactions on the site-specific energetics (including cooperativity effects) of cI and cro repressor binding to the DNA operator sites. The results will provide new insights into the detailed roles of proton binding, salt binding, and the various non-covalent forces responsible for the regulatory interactions. These studies will be extended to a series of mutant repressors that are deficient in cooperativity. We will study the biological role of cooperative interactions between operator-bound repressors by in vitro and in vivo studies of mutant repressors that have altered cooperativity, using a physical-chemical model of gene control that is being developed under this program of research.