PROTEIN-PROTEIN INTERACTIONS IN PROKARYOTIC GENE CONTROL

基因表达的控制依赖于DNA结合蛋白和它们的 互动。这项建议集中在两个特征良好的原核生物 调节因子、lambda抑制子和E.ColiCAP蛋白。长期目标 这项拟议的研究是对(I)的详细分子理解 调节器之间相互作用的性质与一个调节器之间的相互作用有一定距离 另一种是DNA上的，以及(Ii) 激活剂和RNA聚合酶。 当以下两种情况下，Lambda抑制子协同结合到一对操作位点 这些地点是相邻的，就像在自然情况下一样，在人工情况下 将它们分开的构造。在后一种情况下，合作社 相互作用会在DNA中形成一个环。分子 合作结合的基础将是鉴定突变体的遗传筛选 表现出更弱或更强的协作性，并通过隔离 突变体/抑制子对。这一方法将得到网站的补充- 一旦特定残基被牵连，就会发生定向突变。 Lambda抑制因子也能刺激转录。这种激活很可能 涉及DNA=结合阻遏物和RNA聚合酶之间的直接接触。 明确了抑制子的“活化面”。我们建议使用 用遗传和生化方法来鉴定哪个亚基 聚合酶与阻遏物和涉及的特定氨基酸相联系。 在这种互动中。研究CAP和抑制子是否被激活 被分离的聚合酶突变体也会有类似的机制 测试它们对CAP刺激的转录的影响。另外两个 有关激活的问题将得到解决：(I)我们将研究 阻断的基础，在激活时观察到抑制物结合了一些 上游距离与结合在活化点的分子相互作用， (Ii)我们将确定抑制器是否可以远距离激活。 分析原核生物调节蛋白的原理 继续证明对正常和异常控制的研究是有用的 基因表达是许多重要的细胞过程的基础 真核生物，包括胚胎发生和肿瘤发生。

The control of gene expression depends on DNA-bound proteins and their interactions. This proposal focuses on two well-characterized prokaryotic regulators, lambda repressor and E. coli CAP protein. The long term goal of the proposed research is a detailed molecular understanding of (i) the nature of interactions between regulators bound some distance from one another along DNA, and (ii) the nature of the interaction between an activator and RNA polymerase. Lambda repressor binds cooperatively to pair of operator sites both when those sites are adjacent, as in the natural situation, and in artificial constructs where they are separated. In the latter case, the cooperative interaction induces the formation of a loop in the DNA. The molecular basis of cooperative binding will be genetic screens to identify mutants that exhibit either weaker or stronger cooperativity and by isolating mutant/suppressor pairs. This approach will be complemented by site- directed mutagenesis once specific residues have been implicated. Lambda repressor also stimulates transcription. This activation probably involves a direct contact between DNA=bound repressor and RNA polymerase. The 'activation surface' of repressor is well defined. We propose to use both genetic and biochemical approaches to identify which subunit of polymerase is contacted by repressor and the specific amino acids involved in that interaction. To investigate whether CAP and repressor activate by similar mechanisms the polymerase mutants that are isolated will also be tested for their effect on CAP-stimulated transcription. Two additional issues regarding activation will be addressed: (i) We will examine the basis of the block in activation observed when a repressor bound some distance upstream interacts with the molecule bound at the activation site, (ii) We will determine whether repressor can activate from a distance. Principles derived from analysis of prokaryotic regulatory proteins continue to prove useful in the study of normal and abnormal control of gene expression which underlies many important cellular processes in eukaryotes, including embryogenesis and oncogenesis.