CRYSTALLOGRAPHIC STUDIES OF THE LACTOSE REPRESSOR

我们研究的主要目标是了解蛋白质如何 对代谢物做出反应并调节转录。 我们的工作重点 在乳糖操纵子上。 多年来，大肠杆菌的紫胶操纵子 充当基因调控的范例。 紫胶操纵子提供了一种 理解一组结构基因如何发挥作用的最佳模型 根据代谢物的浓度打开或关闭。 操纵子的关键调节成分是 lac 阻遏蛋白。 的 lac 阻遏物，在没有细胞外乳糖的情况下，紧密结合 作用于操纵基因 DNA 并阻止结构基因的转录 乳糖用作生物燃料所需的。 当乳糖为 存在于介质中，阻遏物与操纵子分离 允许结构基因的转录。 构象 抑制子响应结合配体的转变提供了 变构基础。 在上一个资助期内，我们确定了三维 完整的 lac 阻遏蛋白的结构，该 lac 阻遏蛋白与 无偿诱导剂 1-异丙基-β-D-硫代半乳糖苷 (IPTG) 和 lac 结构提供了抑制子的详细结构模型 诱导和抑制状态以及基因的分子基础 监管。 此外，阻遏物的结构提供了 理解丰富的生化和遗传知识的框架 信息。 当前提案的总体目标是建立在 生化、遗传和结构数据以获得更详细的信息 了解阻遏蛋白结合的特异性和结构 变构反应的基础。 使用生物化学和结构方法，我们将探索结构 抑制子功能的基础。 该提案的具体目标是： 阐明 lac 阻遏物及其操纵子之间的相互作用 并确定阻遏蛋白的结合方式是否存在差异 不同的运营商站点；了解突变阻遏蛋白如何结合 与操作员的亲和力增加；以确定结构和 与 DNA 非特异性结合的物理基础；并检查 效应分子变构信号传导的结构基础 作为扰乱信号传导的突变阻遏物。 在所有转录调节因子中，lac 系统是最重要的 彻底研究过。 因此阻遏物及其复合物 操纵基因 DNA 和效应分子具有当代和 对于理解基因调控具有历史重要性。

The major objective of our research is to understand how proteins respond to metabolites and regulate transcription. Our work has focused on the lactose operon. For many years the lac operon of E. coli has served as the paradigm for gene regulation. The lac operon provides one of the best models for understanding how a set of structural genes may be switched on or off depending upon the concentration of metabolites. The key regulatory component of the operon is the lac repressor. The lac repressor, in the absence of extracellular lactose, binds tightly to the operator DNA and prevents transcription of the structural genes required for lactose to be used as a biological fuel. When lactose is present in the medium, the repressor dissociates from the operator allowing transcription of the structural genes. The conformational transitions of the repressor in response to bound ligands provided the basis of allostery. In the previous grant period, we determined the three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer 1-isopropyl-beta-D-thiogalactoside (IPTG) and the lac structures provide a detailed structural model for repressor in the induced and the repressed states and the molecular basis of gene regulation. In addition, the structure of the repressor provides a framework for understanding a wealth of biochemical and genetic information. The overall goal of the current proposal is to build on the biochemical, genetic, and structural data to obtain a more detailed understanding of the specificity of repressor binding and the structural basis for the allosteric response. Using biochemical and structural methods we will explore the structural basis of repressor function. The specific aims of this proposal are to: elucidate the interactions between the lac repressor and its operator and determine if there are differences in the way repressor binds to different operator sites; to understand how mutant repressors can bind to operator with increases affinity; to determine the structural and physical basis for nonspecific binding to DNA; and to examine the structural basis of allosteric signaling by effector molecules as well as mutant repressors that disrupt the signaling. Of all transcriptional regulators the lac system has been the most thoroughly studied. As a consequence the repressor and its complexes with operator DNA and effector molecules have both contemporary and historical importance for the understanding of gene regulation.