STRUCTURE/FUNCTION OF TRP REPRESSOR

控制Trp操纵子在大肠杆菌中的表达是一种遗传范式 微生物代谢的调节。通过行动进行消极控制 色氨酸抑制物对色氨酸可获得性的反应是 实行以监管为主的机制。色氨酸阻遏物是一种DNA结合蛋白 包含螺旋-旋转-螺旋结构基序的转录因子。 DNA结合转录因子调节许多正常和疾病 相关的生物过程。事实上，其中的某些关键事件 发育是含有同源结构域的蛋白质作用的结果。 同源结构域已被证明包含螺旋-转弯-螺旋结构 Motif。此外，细胞调节蛋白如Myc、Fos、Jun 和Myb，以及同源的病毒调节因子可以致癌，如果异常的话 表达的或突变的 我们研究的目标是充分了解分子 DNA结合调节蛋白利用的机制来执行其 生物功能。目前，我们正集中力量建立一个 具有良好特性的模型蛋白，大肠杆菌的色氨酸抑制因子。这个 拟议的研究旨在解决具体的悬而未决的问题 关于色氨酸抑制因子的功能。 之前，我们确定了一种结构域类型，即 NH2-末端臂，参与阻遏物与 运算符形成复合体。尚未在中解析此域 结构研究。我们将进行全面的突变分析 以确定手臂的功能大小 以及手臂氨基酸残基的重要特征。 此外，NH2-终端臂可以调节滑动机构 操作员搜索。我们将测试野生型色氨酸抑制因子和ARM突变体 这一重要机制的抑制者。 我们还将使用一种新开发的基于PCR的技术来测试该模型 建议用“间接读数”来调节Trp的特异性 抑制子/操纵子结合。我们将识别并分离一个 色氨酸抑制物可以高亲和力结合的随机DNA池， 描述每个DNA中的结合位置以及 每个人都有抑制物。 这项工作将增进我们对基本原则的理解 调节转录因子和转录因子相互作用 染色体结合部位。

Control of trp operon expression in E. coli is a paradigm of genetic regulation of microbial metabolism. Negative control through the action of trp repressor in response to tryptophan availability is the predominant regulatory mechanism imposed. trp repressor is a DNA-binding transcription factor that contains the helix-turn-helix structural motif. DNA-binding transcription factors regulate many normal and disease related biological processes. In fact, certain critical events in development result from the action of homeodomain containing proteins. The homeodomain has been shown to contain the helix-turn-helix structural motif. Additionally, cellular regulatory proteins such as Myc, Fos, Jun and Myb, and the cognate viral regulators can be oncogenic if aberrantly expressed or mutated. The goal of our research is a full understanding of the molecular mechanisms utilized by DNA-binding regulatory proteins to perform their biological functions. Currently, we are focusing our efforts on a well-characterized model protein, the trp repressor of E. coli. The proposed research seeks to address specific outstanding questions concerning trp repressor function. Previously, we determined that one type of structural domain, the NH2-terminal arm, is involved in association of the repressor with operator to form complexes. This domain has not been resolved in structural studies. We will perform a comprehensive mutational analysis of the arm domain in order to determine the functional size of the arms and the important characteristics of the arm amino acid residues. Further, the NH2-terminal arms may mediate a sliding mechanism of operator search. We will test the wild type trp repressor and arm mutant repressors for this important mechanism. We will also use a newly developed PCR-based technique to test the model of "indirect readout" proposed to mediate specificity of trp repressor/operator binding. We will identify and isolate all DNAs in a pool of random DNA that trp repressor can bind with high affinity, characterize the binding site within each DNA and the affinity of repressor for each. This work will enhance our understanding of the fundamental principles that mediate interactions between regulatory transcription factors and chromosomal binding sites.