Combinatorial Regulation of Differential Gene Expression in E. coli

大肠杆菌差异基因表达的组合调控

• 批准号: 0215769
• 负责人: Donald Senear
• 金额: $ 52万
• 依托单位: University of California-Irvine
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-15 至 2007-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0215769&HistoricalAwards=false
• 关键词: Combinatorial; Regulation; Differential; Gene; Expression

Coordinate regulation of unlinked genes is fundamental to genetic expression in all organisms. Such regulation relies on the cooperative, site-specific assembly of regulatory, protein-DNA complexes. Combinatorial strategies are common. These involve both widely used transcription factors and also factors that are specific either to the gene, the tissue or cell type, or the development stage. Variable arrays of DNA sites, as are generally found in different genes, assemble different protein-DNA complexes from the same factors. In this manner, unlinked genes can be expressed differentially. Whole programs of gene expression are easily controlled by modulating the activity of only the specific factors. This project addresses the combinatorial mechanism of differential gene expression in the E. coli CytR regulon. This gene family consists of nine unlinked transcription units that are regulated coordinately by the interplay of just two regulatory proteins. One is the cyclic-AMP receptor protein (CRP) a widely used transcriptional activator in bacteria; the other, CytR, is a regulon-specific bacterial repressor. CytR is also a member of the LacI family of homologous repressors. A key feature of the regulation is that the various cistrons differ from one another in extents of activation, repression and induction. This differential regulation is achieved by nesting levels of local repression, mediated by CytR, on the global regulation mediated by CRP. An unusual feature of most CytR-regulated promoters is that they contain two CRP sites. These activate transcription via different mechanisms. CytR and CRP bind cooperatively to DNA, as a result of direct protein-protein interactions. The importance of cooperativity is underscored by the fact that induction occurs when CytR binds cytidine because the CytR-CRP cooperativity is lost and despite no affect on intrinsic CytR binding to DNA. Unlike most bacterial repressors, CytR does not repress transcription independently; instead it acts by modulating the CRP-mediated activation. It does so by interacting with DNA-bound CRP to control the interactions between activating regions on CRP and the bacterial RNA Polymerase. The objectives of the research are: 1) to understand how the different structures of CytR-regulated promoters mediate different patterns of cooperative interactions between CRP and CytR; and 2) to understand the role of CytR-CRP cooperativity in controlling activation and repression of RNAP at different promoters, using both site occlusion and allosteric mechanisms. To accomplish these goals a comparison of the interactions among CRP, CytR and the four class III CytR-regulated promoters will be made using DNase footprinting and gel mobility shift assays. The role of different binding modes of CytR and different arrangements of CytR and CRP binding sites will be assessed systematically using artificial promoters. These will allow independent controls on the structure of the CytR operator and locations of regulatory sites. These data will be combined with results of both steady state and pre-steady state assays of transcription initiation to develop a comprehensive kinetic model of differential gene regulation. CytR and CRP mutants that are defective either in their mutual interaction or in their interaction with RNAP will be used to assess the role of the interaction between CytR and CRP bound to CRP2 in modulating the class II mediated activation.This project is supported by the Biochemistry of Gene Expression Program and the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.

未连锁基因的协调调控是所有生物体基因表达的基础。 这种调节依赖于调节性蛋白质-DNA 复合物的协作性、位点特异性组装。 组合策略很常见。 这些既涉及广泛使用的转录因子，也涉及基因、组织或细胞类型或发育阶段特有的因子。 DNA 位点的可变阵列通常存在于不同的基因中，它们从相同的因子组装不同的蛋白质-DNA 复合物。 以这种方式，未连锁的基因可以差异表达。 通过仅调节特定因子的活性，可以轻松控制基因表达的整个程序。 该项目研究了大肠杆菌 CytR 调节子中差异基因表达的组合机制。 该基因家族由九个不相连的转录单位组成，这些单位仅通过两个调节蛋白的相互作用来协调调节。 一种是环磷酸腺苷受体蛋白（CRP），一种在细菌中广泛使用的转录激活剂；另一个是 CytR，是一种调节子特异性细菌阻遏蛋白。 CytR 也是同源阻遏蛋白 LacI 家族的成员。该调节的一个关键特征是各种顺反子在激活、抑制和诱导的程度上彼此不同。 这种差异性调节是通过 CytR 介导的局部抑制嵌套在 CRP 介导的全局调节上来实现的。 大多数 CytR 调节的启动子的一个不寻常的特征是它们包含两个 CRP 位点。 它们通过不同的机制激活转录。 CytR 和 CRP 协同结合 DNA，这是蛋白质与蛋白质直接相互作用的结果。 当 CytR 结合胞苷时发生诱导，这一事实强调了协同性的重要性，因为 CytR-CRP 协同性丢失，尽管对内在 CytR 与 DNA 的结合没有影响。 与大多数细菌阻遏蛋白不同，CytR 并不独立地抑制转录；相反，它通过调节 CRP 介导的激活来发挥作用。 它通过与 DNA 结合的 CRP 相互作用来控制 CRP 激活区域和细菌 RNA 聚合酶之间的相互作用来实现这一点。 研究目的是：1）了解CytR调控启动子的不同结构如何介导CRP和CytR之间不同的合作相互作用模式； 2) 使用位点封闭和变构机制了解 CytR-CRP 协同性在不同启动子处控制 RNAP 激活和抑制中的作用。 为了实现这些目标，将使用 DNase 足迹和凝胶迁移率变化测定来比较 CRP、CytR 和四个 III 类 CytR 调节启动子之间的相互作用。 将使用人工启动子系统地评估 CytR 的不同结合模式以及 CytR 和 CRP 结合位点的不同排列的作用。 这些将允许对 CytR 运营商的结构和监管站点的位置进行独立控制。 这些数据将与转录起始的稳态和稳态前测定结果相结合，以开发差异基因调控的综合动力学模型。 在相互相互作用或与 RNAP 相互作用方面有缺陷的 CytR 和 CRP 突变体将用于评估 CytR 和与 CRP2 结合的 CRP 之间的相互作用在调节 II 类介导的激活中的作用。该项目由生物化学支持生物科学局分子和细胞生物科学部的基因表达计划和分子生物物理学计划。