GENETIC ANALYSIS OF REPRESSOR/DNA RECOGNITION

我们将采用分子遗传学的方法来理解结构和 大肠杆菌色氨酸受体功能，一个小的(107个残基)， 稳定的蛋白质，与色氨酸结合形成活性抑制物(DNA- 约束性)复合体。我们正在用每一个突变的非抑制子 2,033个可能的单一氨基酸变化，并分析它们的表型 活着。从这个含有单一氨基酸的突变蛋白质集合中 变化，我们应该能够确定抑制物的哪些残基是 对折叠和二聚化至关重要，对结合辅阻遏子至关重要 (色氨酸)，以及结合DNA。此外，这些基因的表型 单个更改将为我们提供一个数据库来检查各种 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，我们还将制作多个Trp阻滞剂，以检查各种 蛋白质结构和功能的分子细节方面。这些 包括多个突变体来研究色氨酸和其他 吲哚类化合物作为辅抑制物使用时间分辨 荧光和核磁共振光谱学，研究DNA结合，并研究 上下文中的不寻常结构(例如，均聚α-螺旋) 这种小的原核蛋白。我们还将合成共价色氨酸 抑制物，用光谱学检查色氨酸结合口袋 更详细的方法。 小尺寸和稳定性的结构很好地表征 蛋白质、色氨酸受体及其相互作用的多面性 通过辅抑制子和DNA，使trpr基因成为非常有吸引力的选择 详细的基因分析。突变型阻抑因子Will的生化研究 为我们提供了关于该结构的无与伦比的丰富信息 和单一调节蛋白的功能。了解 DNA结合蛋白的结构与功能的关系是 对我们理解基因表达调控的核心是 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.