CRYSTALLOGRAPHIC STUDIES OF THE LACTOSE REPRESSOR

乳糖抑制剂的晶体学研究

• 批准号: 3303816
• 负责人: MITCHELL LEWIS
• 金额: $ 24.42万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3303816
• 关键词: DNA; DNA binding protein; X ray crystallography; chemical binding; computer program /software; computer simulation; conformation; crystallization; genetic operator element; genetic regulation; high performance liquid chromatography; interferometry; isomorphous substitution; lac operon; lactose; molecular site; mutant; nucleic acid chemical synthesis; physical model; protein purification; protein structure function; stoichiometry; structural biology; synthetic nucleotide; transcription factor

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.

乳糖操纵子已成为研究基因调控的范例。 的 lac阻遏物及其复合物的x射线晶体学分析 与效应分子和乳糖操纵基因DNA将提供结构 了解基因调控的平台。 我们的主要兴趣是 了解阻遏物如何识别特定的序列， 操纵基因 同样重要的是建立结合位点， 诱导剂和抗诱导剂，然后，确定这些效应物 控制DNA结合。 这些结构还将提供信息 关于变构的结构方面。 基因数据描绘了 阻遏物的四个功能区。 特定的点突变体具有 分离出的表型在以下方面有缺陷：操纵基因DNA 结合、诱导物结合、信号传递和四元 协会. 我们的主要重点是确定三维 lac阻遏物的结构，以实验阐明其结构 效应物结合，并确定共晶的结构， 具有对称21个碱基对Lac操纵子的Lac阻遏物。 结构 的lac阻遏物将使我们能够解决这样的问题：（1）如何 阻遏物是否识别DNA上的特定位点，（2）如何识别 诱导剂和抗诱导剂影响阻遏物的构象， 最终（3）结构与变构机制有何关系 用于调节基因表达。 已经分析了超过4000个单个氨基酸替换， 表型 因此，出现了更多的功能变体 在乳糖阻遏物中的氨基酸取代产生的比在任何 其他蛋白质包括血红蛋白。 定点突变， 这个项目是所有蛋白质中最全面的。 分析 关于三级和四级结构的突变体， 将是这个项目的结果将提供令人兴奋的见解， 蛋白质折叠 随着三维结构的掌握， lac阻遏物将是研究蛋白质折叠最完整的系统。 天然lac阻遏物的结构研究 阻遏物-DNA复合物的复合物和共晶体将提供长的 期待结构框架更完整地描述乳糖操纵子- - 原型基因调控系统。