Specificity & Regulatory Properties of trp Repressor

这是一项研究蛋白质对核酸的特异性识别的分子基础，以及涉及它们在体内和体外的调节相互作用的因素的建议。这项工作将集中在具有良好结构特征的大肠杆菌色氨酸抑制物上，并将集中在五个方面。1.将深入研究操作者DNA的结构/功能关系，包括它们在识别过程中的作用。将合成代表操纵子的寡核苷酸，以及组成相同但序列无关的对照DNA。这些DNA的平均溶液构象将用圆二色谱(CD)推迟挖掘。这些DNA的螺旋-线圈转变的热力学将用CD来确定，并将结果与在阻滞剂存在下获得的结果进行比较。2.将开发新的可靠的、定量的色氨酸抑制物结合反应的分析方法。首先，将确定蛋白质结合是否会引起DNA CD光谱的变化；这种方法可能只适用于非特异性结合。为了检测特异性结合，荧光探针将被附着在DNA上，在那里它们可能被结合的蛋白质猝灭，而不会对其结合产生很大的干扰。3.色氨酸阻遏物与DNA结合的亲和力和特异性将在用于络合物结晶的确切条件下确定。结果应该确定这些条件是否与操作者特定的结合兼容，从而解决由晶体结构引起的主要争议。4.对抑制物体外结合反应的生化和热力学特征进行全面的分析。将测量与特定和非特定DNA以及色氨酸结合的盐浓度和温度依赖关系，并与已知的蛋白质结构相关。我们将被用来确定有序-无序转变是否可以解释在温度研究中发现的DNA结合的大CP。5.进行体内和体外实验，以确定Trp抑制基因转录调控的详细分子机制。抑制子对RNA聚合酶的影响在三个已知的调控位点是否相同，以及聚合酶机制中的哪个步骤在每个位点受到影响的问题将被调查。三种操纵子在体内的亲和力将被比较，并将确定抑制子和聚合酶是否通过通讯来调节彼此与DNA的相互作用。

This is a proposal to study the molecular basis for the specific recognition of nucleic acids by proteins, and the factors involved their regulatory interactions, both in vivo and in vitro. The work will focus on the structurally well- characterized trp repressor of E. coli, and will be concentrated in five areas. 1. A thorough study of operator DNA structure/function relations will be made including their role in the recognition process. An oligonucleotide representing the operator will be synthesized, along with a control DNA of identical composition but unrelated sequence. The average solution conformation of these DNAs will be defer mined using circular dichroism (CD). The thermodynamics of the helix-coil transition for these DNAs will be determined using CD, and the results will be compared to those obtained in the presence of the repressor. 2..Development of new reliable, quantitative assays for the binding reactions of trp repressor will be undertaken. First, it will be ascertained whether protein binding causes a change in the CD spectrum of the DNA; this assay is likely to be suitable only for nonspecific binding. In an effort to assay specific binding, fluorescent probes will be attached to the DNA at sites where they may be quenched by the bound protein without interfering greatly with its binding. 3. The affinity and specificity of trp repressor for binding to DNA will be determined under the exact conditions that have been used for crystallization of the complex. The results should establish whether these conditions are compatible with operator-specific binding, and would thus resolve a major controversy engendered by the crystal structure. 4. A complete analysis of the biochemical and thermodynamic characteristics of the in vitro binding reactions of the repressor will be made. Salt concentration and temperature dependence for binding to specific and nonspecific DNA and to tryptophan will be measured, and related to what is known about the structure of the protein. We will be used to establish whether an order-disorder transition explains the large CP for DNA binding found in the temperature studies. 5. In vivo and in vitro experiments will be carried out to determine the detailed molecular mechanism of transcriptional control by trp repressor. The question of whether the effect of repressor on RNA polymerase is the same at the three known sites of regulation, and which step in the polymerase mechanisms is affected at each site will be investigated. The affinities of the three operators in vivo will be compared, ant it will be determined if repressor and polymerase communicate to modulate each other's interactions with the DNA.