CRYSTALLOGRAPHIC STUDIES OF THE LACTOSE REPRESSOR

我们研究的主要目标是了解蛋白质是如何 对代谢物作出反应并调节转录。我们的工作重点是 在乳糖操纵子上。多年来，大肠杆菌的乳胶操纵子 作为基因调控的范例。乳胶操纵子提供了一种 了解一组结构基因如何 根据代谢物的浓度打开或关闭。 操纵子的关键调控成分是lac抑制因子。这个 在没有细胞外乳糖的情况下，乳糖抑制物紧密结合 结合操作者DNA，阻止结构基因转录 将乳糖用作生物燃料所需的。当乳糖是 存在于介质中的抑制子与操作者解离 允许结构基因的转录。构象 抑制物对结合配体的反应转变提供了 变构的基础。 在之前的资助期间，我们确定了三维 完整的乳胶抑制物的结构，乳胶抑制物结合到 无偿诱导剂1-异丙基-β-D-硫代半乳糖苷(IPTG)和紫胶 结构为抑制器提供了一个详细的结构模型 基因的诱导和抑制状态及其分子基础 监管。此外，抑制子的结构提供了一个 了解丰富的生物化学和遗传学的框架 信息。目前提案的总体目标是在 生化、遗传和结构数据，以获得更详细的 对抑制子结合的特异性和结构的理解 变构反应的基础。 利用生化和结构方法，我们将探索结构 抑制器功能的基础。这项建议的具体目的是： 阐明lac抑制子与其操纵子之间的相互作用 并确定抑制物结合的方式是否存在差异 不同的操作入口；了解突变抑制物如何结合 以增加亲和力的运算符；确定结构和 与DNA的非特异性结合的物理基础；并检查 效应分子的变构信号的结构基础 作为干扰信号的突变抑制子。 在所有转录调控因子中，lac系统是最多的。 彻底研究过。因此，抑制物及其复合体 与操作者DNA和效应器分子同时具有当代和 对理解基因调控的历史意义。

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.