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; 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.

未链接基因的坐标调节是所有生物体中遗传表达的基础。 这种调节依赖于调节性，蛋白质-DNA复合物的合作，特定地点组装。 组合策略很常见。 这些涉及广泛使用的转录因子，也涉及特定于基因，组织或细胞类型或发育阶段的因素。 DNA位点的可变阵列（通常在不同基因中发现）从相同因素组装出不同的蛋白-DNA复合物。 以这种方式，未链接基因可以差异表达。 基因表达的整个程序仅通过调节特定因素的活性而容易控制。 该项目解决了大肠杆菌Cytr调节中差异基因表达的组合机制。 该基因家族由九个未连接的转录单元组成，这些单元仅由仅两个调节蛋白的相互作用协调。 一种是细菌中广泛使用的转录激活剂的环状受体蛋白（CRP）。另一个是Cytr，是一种调节特异性细菌阻遏物。 CYTR还是LACI同源阻遏物家族的成员。该法规的一个关键特征是，各种cistrons在激活，抑制和诱导的范围内彼此不同。 通过CYTR介导的局部抑制作用的嵌套水平，在由CRP介导的全球调节中实现了这种差异调节。 大多数CYTR调节的启动子的一个不寻常特征是它们包含两个CRP位点。 这些通过不同的机制激活转录。 由于直接蛋白 - 蛋白质相互作用，CYTR和CRP与DNA结合。 当Cytr结合胞丁时，由于Cytr-CRP协作丢失并且尽管对固有的cytr结合与DNA没有影响，因此同时性的重要性强调了诱导发生。 与大多数细菌阻遏物不同，CYTR不会独立抑制转录。相反，它通过调节CRP介导的激活来起作用。 它通过与结合DNA的CRP相互作用来控制CRP激活区域与细菌RNA聚合酶之间的相互作用。 研究的目标是：1）了解Cytr调节的启动子的不同结构如何介导CRP和CYTR之间的不同合作相互作用模式； 2）了解使用位点遮挡和变构机制，了解Cytr-CRP协作在控制不同启动子处的RNAP激活和抑制中的作用。 为了实现这些目标，将使用DNASE足迹和凝胶移动性转移分析进行比较CRP，CYTR和四个III CYTR调节的启动子之间的相互作用。 CYTR的不同结合模式以及CYTR和CRP结合位点不同排列的作用将使用人工启动子进行系统评估。 这些将允许对CYTR操作员的结构和调节位置的位置进行独立控制。 这些数据将与转录启动的稳态和稳态测定的结果相结合，以开发出差异基因调控的全面动力学模型。 Cytr和CRP突变体在相互相互作用或与RNAP的相互作用中存在有缺陷的情况，将使用与CRP2结合在调节II类介导的激活中的相互作用的作用。该项目由基因表达程序的生物化学疗法和分子生物学对生物学的生物科学进行了生物学分析，并在生物科学中进行了生物学分析。