Cooperative Interactions of Gene-Regulatory Proteins

基因调控蛋白的协同相互作用

• 批准号: 8918670
• 负责人: Michael Fried
• 金额: $ 5.8万
• 依托单位: University of Texas Health Science Center San Antonio
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1991-07-01
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8918670&HistoricalAwards=false
• 关键词: Cooperative; Interactions; Gene; Regulatory; Proteins

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. //

本研究的目的是鉴定蛋白质-DNA， 蛋白质-蛋白质相互作用调节的组装 原核转录起始复合物，以及 获得和了解他们在控制其 活动 对E.大肠杆菌RNA聚合酶， 乳糖（lac）阻遏物和环AMP受体蛋白（CAP） 将研究乳糖启动子-操纵基因区域。 这些蛋白质的个体活动 该地区一直是大量的主题， 研究，并具有良好的特性。 缺少的是什么 现在是了解它们如何相互作用， 转录。 为了解决这一关键问题，研究将 进行到：1.确定每种蛋白质的作用 对lac启动子结合亲和力、化学计量和 相互作用的每个其他网站; 2.测量 每种蛋白质对结合和解离的影响 每个其他人的动力学; 3.表征 每个含有RNA的复合物的转录活性 （a）异构化状态-是否 “开放”或“封闭”;（B）其失败的引发动力学;（c） 流产与生产性启动的比率和（d） 两个转录起始点的比率 重叠lac启动子（P1和P2）。 4.确定 启动子外调控序列对 CAP、lac复合物的组成、稳定性和活性 阻遏物和RNA聚合酶在体外的乳糖启动子， 在体内诱导转录。 因为每种蛋白质都至少有两个高亲和力结合 lac启动子内的位点，几种不同的结合 排列和转录活性状态可能存在 对于给定的结合化学计量。 因此，具体 目标1和3将以协调一致的方式实现， 允许蛋白质化学计量、结合 每个复合物的位点占有率和转录活性。//