CRYSTALLOGRAPHIC STUDIES OF THE LACTOSE REPRESSOR

乳胶操纵子已经成为研究基因调控的范例。这个 紫胶阻滞剂及其络合物的X射线结晶学分析 通过效应器分子和lac操纵者DNA将提供结构 了解基因调控的平台。我们的主要兴趣是 了解抑制器如何识别特定的序列 操作者DNA。同样重要的是要建立与 诱导剂和抗诱导剂，然后，确定这些效应器是如何 控制DNA结合。这些结构还将提供信息 关于变构的结构方面。基因数据已经描绘出 抑制因子的四个功能区。特异点突变体有 被分离出有缺陷的表型：操作员DNA 结合、诱导剂结合、信号传递和四元 联想。我们的主要关注点将是确定三维 乳胶抑制物的结构，从实验上阐明其结构 效应器结合，并确定共晶体的结构 带有对称的21个碱基对的Lac操纵子的Lac抑制子。该结构 将使我们能够解决如下问题：(1)如何 抑制子识别DNA上的特定位置吗？(2)如何 诱导剂和反诱导剂影响抑制子的构象，并且 最终(3)结构如何与变构机制相关 用来调节基因表达。 已经分析了4000多个单一氨基酸的替代 表型。因此，出现了更多的功能变体 由紫胶抑制物中的氨基酸取代产生的 包括血红蛋白在内的其他蛋白质。该位点定向突变于 这个项目是所有蛋白质中最全面的。一种分析 关于三元和四元结构的突变体 将是这个项目的结果将提供令人兴奋的洞察 蛋白质折叠。随着三维结构的良好掌握， Lac抑制物将是研究蛋白质折叠的最完整的系统。 天然紫胶抑制因子--抑制因子诱导剂的结构研究 抑制子-DNA复合体和共晶体将提供长时间的 等待更完整地描述乳胶操纵子的结构框架- --原型基因调控系统。

The lac operon has served as the paradigm to study gene regulation. The x-ray crystallographic analyses of the lac repressor and its complexes with effector molecules and lac operator DNA will provide the structural platform for understanding gene regulation. Our primary interest is to understand how the repressor recognizes the specific sequence of the operator DNA. It is also important to establish the binding sites for inducers and anti-inducers, and then, to establish how these effectors control DNA binding. These structures will also provide information about the structural aspects of allostery. Genetic data has delineated four functional regions of the repressor. Specific point mutants have been isolated with phenotypes that are defective in: operator DNA binding, inducer binding, signal transmission, and quaternary associations. Our main focus will be to determine the 3-dimensional structure of the lac repressor, to experimentally elucidate the structure the effectors binding, and determine the structure of cocrystals of the lac repressor with a symmetric 21 base pair lac operator. The structure of the lac repressor will allow us to address such questions as (1) how does the repressor recognize specific sites on the DNA, (2) how do inducers and anti-inducers affect the conformation of the repressor, and ultimately (3) how does the structure relate to the allosteric mechanism used to regulate gene expression. Over four thousand single amino acid substitutions have been analyzed for phenotype. As a consequence, there have been more functional variants produced by amino acid substitutions in the lac repressor than in any other protein including hemoglobin. The site directed mutagenesis on this project is the most comprehensive of all proteins. An analysis of the mutants with respect to the tertiary and quaternary structures that will be the result of this project will provide exciting insights into protein folding. With the three dimensional structure well in hand, the lac repressor will be the most complete system to study protein folding. The structural studies of the native lac repressor, the repressor-inducer complex and cocrystals of the repressor-DNA complex will provide a long awaited structural framework to describe more completely the lac operon - - the prototypical gene regulation system.