GENETIC ANALYSIS OF REPRESSOR/DNA RECOGNITION

我们将采用分子遗传学方法来了解其结构， 大肠杆菌Trp脱辅阻遏物的功能，一个小的（107个残基）， 一种稳定的蛋白质，与色氨酸结合形成活性阻遏物（DNA- 结合）复合物。 我们正在用每一种基因制造突变的去阻遏蛋白。 2，033个可能的单一氨基酸变化，并分析其表型， vivo. 从这些单一氨基酸的突变蛋白质中 改变，我们应该能够确定哪些残基的脱辅基阻遏物是 对于折叠和二聚化，对于结合辅阻遏物 （色氨酸）和用于结合DNA。 此外，这些表型 单一的变化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 组合盒式诱变和丙氨酸扫描。 此外，我们还将制备多种Trp去辅阻遏物， 蛋白质结构和功能的分子细节方面。 这些 包括多个突变体，以研究色氨酸和其他 作为辅阻遏物的吲哚衍生物， 荧光和核磁共振光谱，研究DNA结合，并研究 异常结构（例如，均聚物α-螺旋） 这种小的原核蛋白质。 我们还将合成共价色氨酸 阻遏物，通过光谱分析来检查色氨酸结合口袋 方法更详细。 小尺寸和稳定性的结构良好的特点 蛋白质，色氨酸脱辅阻遏物，以及其相互作用的多方面性质 与辅阻遏物和DNA，使trpR基因是一个非常有吸引力的选择， 详细的基因分析 突变型脱辅基阻遏物的生化研究将 为我们提供了无与伦比的丰富信息 和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 这是我们理解基因表达调控的核心， 分子水平。

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.