CRYSTALLOGRAPHIC STUDIES OF THE LACTOSE REPRESSOR

我们研究的主要目的是了解蛋白质 对代谢物作出反应并调节转录。 我们的工作重点是 乳糖操纵子上 多年来，E.大肠杆菌有 作为基因调控的范例。 乳糖操纵子提供了一个 了解一组结构基因如何 根据代谢物的浓度打开或关闭。 操纵子的关键调节组分是乳糖阻遏物。 的 在没有胞外乳糖的情况下，lac阻遏物紧密结合 并阻止结构基因的转录 乳糖被用作生物燃料所需的。 当乳糖 当阻遏物存在于培养基中时， 允许结构基因的转录。 构象 阻遏物响应于结合配体的转变提供了 变构的基础 在上一个赠款期间，我们确定了三维 完整的lac阻遏物的结构，lac阻遏物结合到 一种无意义的诱导剂1-异丙基-β-D-硫代半乳糖苷（IPTG）， 结构提供了一个详细的结构模型，阻遏物在 基因的诱导和阻遏状态以及基因表达的分子基础 调控 此外，阻遏物的结构提供了一种 理解大量生物化学和遗传学 信息. 本提案的总体目标是在 生物化学，遗传学和结构数据，以获得更详细的 了解阻遏物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 阻遏物功能的基础。 这项建议的具体目标是： 阐明lac阻遏子和其操纵子之间的相互作用 并确定是否有不同的方式阻遏物结合， 不同的操纵基因位点;了解突变阻遏物如何结合 以增加亲和力;以确定结构和 与DNA非特异性结合的物理基础;并检查 效应分子变构信号的结构基础 突变阻遏物破坏信号传导。 在所有的转录调节子中，lac系统是最重要的。 彻底研究。 因此，阻遏物及其复合物 与操纵基因DNA和效应分子同时存在， 对理解基因调控具有历史意义。

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.