GENETIC ANALYSIS OF REPRESSOR DNA RECOGNITION

抑制子 DNA 识别的遗传分析

• 批准号: 2177305
• 负责人: PHILIP A YOUDERIAN
• 金额: $ 3.33万
• 依托单位: UNIVERSITY OF IDAHO
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2177305
• 关键词: DNA binding protein; Escherichia coli; bacterial genetics; binding proteins; dimer; fluorescence spectrometry; gene expression; gene mutation; genetic mapping; molecular cloning; nuclear magnetic resonance spectroscopy; nucleic acid sequence; protein folding; protein structure function; temperature sensitive mutant; transcription factor; tryptophan

We will take a molecular genetic approach to understand the structure and function of Escherichia coli Trp aporepressor, a small (107 residue), stable protein that binds tryptophan to form an active repressor (DNA- binding) complex. We are making mutant aporepressors with each of the 2,033 possible single amino acid changes, and analyzing their phenotypes in vivo. From this collection of mutant proteins with single amino acid changes, we should be able to define which residues of aporepressor are critical for folding and dimerization, for binding corepressor (tryptophan), and for binding DNA. In addition, the phenotypes of these single changes will provide us with a database to examine various strategies for the analysis of protein structure and function, including combinatorial cassette mutagenesis and alanine scanning. In addition, we will make multiple Trp aporepressors to examine various aspects of protein structure and function in molecular detail. These include multiple mutants to study the binding of tryptophan and other indole derivatives that act as corepressors using time-resolved fluorescence and NMR spectroscopy, to study DNA binding, and to study unusual structures (e.g., homopolymeric alpha-helices) within the context of this small prokaryotic protein. We will also synthesize covalent Trp repressor, to examine the tryptophan binding pocket by spectroscopic methods in greater detail. The small size and stability of the structurally well-characterized protein, Trp aporepressor, and the multi-faceted nature of its interactions with corepressors and DNA, make the trpR gene a very attractive choice for detailed genetic analysis. Biochemical study of mutant aporepressors will provide us with an unparalleled wealth of information about the structure and function of a single regulatory protein. Understanding the relationship between the structure and function of DNA-binding proteins is central to our understanding of the regulation of gene expression at the molecular level.

我们将采用分子遗传学方法来了解结构和 大肠杆菌TRP AporePressor的功能，一个小（107个残基）， 稳定的蛋白质结合色氨酸形成活性抑制剂（DNA- 结合）复合物。 我们正在用每个 2,033个可能的单个氨基酸会改变，并分析其表型 体内。 从这个带有单氨基酸的突变蛋白的集合中 更改，我们应该能够定义哪些AporePressor的残基是 对于折叠和二聚化至关重要，对于绑定Corepressor （色氨酸），用于结合DNA。 另外，这些表型 单个更改将为我们提供一个数据库，以检查各种 分析蛋白质结构和功能的策略，包括 组合盒诱变和丙氨酸扫描。 此外，我们将制作多个TRP AporePressor来检查各种 蛋白质结构和功能的各个方面。 这些 包括多个突变体来研究色氨酸和其他突变体 使用时间分辨的吲哚衍生物充当核压剂 荧光和NMR光谱，研究DNA结合并研究 不寻常的结构（例如均聚物α-螺旋）在上下文中 这种小核蛋白。 我们还将合成共价trp 抑制剂，检查色氨酸结合口袋通过光谱镜检查 方法更详细。 结构良好的尺寸和稳定性较小 蛋白质，trp aporepressor及其相互作用的多方面性质 使用Corepressor和DNA，使TRPR基因成为非常吸引人的选择 详细的遗传分析。 突变体压力器的生化研究将 为我们提供有关结构的无与伦比的丰富信息 单一调节蛋白的功能。 了解 DNA结合蛋白的结构和功能之间的关系是 我们理解基因表达调节的核心 分子水平。