Cooperative Interactions of Gene-Regulatory Proteins

本研究的目的是确定蛋白质- dna和蛋白质-蛋白质相互作用，调节原核生物转录起始复合物的组装，并获得和理解它们在控制其活性中的作用。研究了大肠杆菌RNA聚合酶、乳糖抑制因子和环AMP受体蛋白（CAP）在乳糖启动子-操作子区域的功能。这些蛋白质在这一区域内的个体活动已经成为大量研究的主题，并得到了很好的表征。目前缺乏的是对它们如何相互作用来调节转录的理解。为了解决这一关键问题，将进行以下研究：确定每种蛋白质对lac启动子的结合亲和力、化学计量学和相互作用位点的影响；2. 测量每种蛋白质对其他蛋白质的结合和解离动力学的影响；3. 描述每个含有RNA聚合酶的复合物的转录活性：(a)异构化状态-无论是“开放”还是“封闭”；(b)流产起始动力学；(c)在两个重叠的lac启动子（P1和P2）上，每个启动子的转录启动率。4. 确定启动子外调控序列对体外lac启动子上CAP、lac抑制因子和RNA聚合酶复合物的组成、稳定性和活性的影响，以及对体内转录诱导的影响。由于每个蛋白在lac启动子内至少有两个高亲和力的结合位点，因此对于给定的结合化学计量，可能存在几种不同的结合排列和转录活性状态。因此，具体目标1和3将以协调一致的方式进行，允许每个复合物的蛋白质化学计量学，结合位点占用和转录活性的相关性。//

The objective of this research is to identify protein-DNA and protein-protein interactions that regulate the assembly of the prokaryotic transcriptional initiation complex, and to gain and understanding of their roles in the control of its activity. The functions of the E. coli RNA polymerase, lactose (lac) repressor and cyclic AMP receptor protein (CAP) at the lactose promoter-operator region will be investigated. The individual activities of these proteins within this region have been the subjects of a significant amount of research, and are well characterized. What is lacking at present is an understanding of how they interact to regulate transcription. To address this crucial issue, studies will be carried out to: 1. determine the effects of each protein on the lac promoter binding affinities, stoichiometries and sites of interaction of each of the others; 2. measure the effects of each protein on the association and dissociation kinetics of each of others; 3. characterize the transcriptional activity of each complex containing RNA polymerase in terms of: (a) isomerization state- whether "open" or "closed"; (b) its abortive initiation kinetics; (c) the ratio of abortive to productive initiation and (d) the ratio of transcriptional initiation at each of the two overlapping lac promoters (P1 and P2). 4. determine the effects of extra-promoter regulatory sequences on the composition, stability and activity of complexes of CAP, lac repressor and RNA polymerase at the lac promoter in vitro and on the induction of transcription in vivo. Because each protein has at least two high affinity binding sites within the lac promoter, several different binding arrangements and transcriptional activity states may exist for a given binding stoichiometry. For this reason, specific aims 1 and 3 will be carried out in a concerted manner, allowing correlation of the protein stoichiometry, binding site occupancy and transcriptional activity of each complex. //