Combinatorial Regulation of Differential Gene Expression in E. coli

非连锁基因的协调调控是所有生物体基因表达的基础。这种调控依赖于调控蛋白-DNA复合体的合作、特定部位的组装。组合策略很常见。这些既包括广泛使用的转录因子，也包括特定于基因、组织或细胞类型或发育阶段的因子。不同基因中普遍存在的DNA位点的不同排列，由相同的因子组装成不同的蛋白质-DNA复合体。通过这种方式，未连接的基因可以差异表达。通过只调节特定因子的活性，就可以很容易地控制整个基因表达程序。本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。这个基因家族由9个未连接的转录单位组成，它们由仅两个调控蛋白的相互作用协调调节。一种是环-AMP受体蛋白(CRP)，一种在细菌中广泛使用的转录激活因子；另一种是CytR，它是一种调节子特异性的细菌抑制因子。CytR也是LacI同源抑制因子家族的成员。该调控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各不相同。这种不同的调控是通过嵌套水平的局部抑制来实现的，这种局部抑制是由CytR介导的，对由CRP介导的全球调控起作用。大多数由CytR调控的启动子有一个不同寻常的特点，那就是它们含有两个CRP位点。它们通过不同的机制激活转录。由于蛋白质与蛋白质的直接相互作用，细胞色素R和C反应蛋白协同结合到DNA上。当CytR与胞苷结合时发生诱导，这一事实突显了协同性的重要性，因为CytR-CRP的协作性丢失，尽管不影响CytR与DNA的内在结合。与大多数细菌抑制物不同，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上活化区和细菌RNA聚合酶之间的相互作用来实现这一点。本研究的目的是：1)了解CytR调控的启动子的不同结构如何调节CRP和CytR之间不同的协同作用模式；2)了解CytR-CRP的协同作用在控制不同启动子上RNAP的激活和抑制中的作用，包括位点阻断和变构机制。为了实现这些目标，将使用DNA酶足迹和凝胶迁移率改变分析来比较CRP、CytR和四个III类CytR调节的启动子之间的相互作用。将利用人工启动子系统地评估不同的细胞色素R结合方式以及不同排列的细胞色素R和C反应蛋白结合位点的作用。这将允许对CytR运营商的结构和监管地点的位置进行独立控制。这些数据将与稳定状态和稳定前转录启动分析的结果相结合，以开发差异基因调控的综合动力学模型。在相互作用或与RNAP的相互作用中有缺陷的CytR和CRP突变体将被用来评估与CRP2结合的CytR和CRP之间的相互作用在调节第二类介导的激活中的作用。该项目得到了生物科学局分子和细胞生物科学部的基因表达生物化学计划和分子生物物理计划的支持。

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.