Combinatorial Regulation of Differential Gene Expression in E. coli

非连锁基因的协调调节是所有生物基因表达的基础。这种调节依赖于调节蛋白- dna复合物的协同、位点特异性组装。组合策略很常见。这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。DNA位点的可变排列，通常在不同的基因中发现，从相同的因子中组装不同的蛋白质-DNA复合物。通过这种方式，非连锁基因可以有差异地表达。基因表达的整个程序很容易通过调节特定因子的活性来控制。本项目研究大肠杆菌CytR调控中差异基因表达的组合机制。该基因家族由9个非连锁转录单位组成，这些单位由两种调节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剂；另一种是CytR，是一种特定于规则的细菌抑制因子。CytR也是同源抑制因子LacI家族的一员。调节的一个关键特征是不同的顺子在激活、抑制和诱导的程度上彼此不同。这种差异调节是通过CytR介导的局部抑制水平嵌套在CRP介导的全局调节上实现的。大多数cytr调控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，作为直接的蛋白质-蛋白质相互作用的结果。协同性的重要性被这样一个事实所强调，即当CytR与胞苷结合时，诱导发生，因为CytR- crp的协同性丧失，尽管对CytR与DNA的内在结合没有影响。与大多数细菌抑制因子不同，CytR并不独立抑制转录；相反，它通过调节crp介导的激活来起作用。它通过与dna结合的CRP相互作用来控制CRP上的激活区与细菌RNA聚合酶之间的相互作用。本研究的目的是：1)了解不同结构的CytR调控启动子如何介导CRP与CytR之间不同的协同相互作用模式；2)了解CytR-CRP协同作用在控制不同启动子上RNAP的激活和抑制中的作用，包括位点闭塞和变构机制。为了实现这些目标，我们将使用dna酶足迹和凝胶迁移试验来比较CRP、CytR和四种CytR调控的III类启动子之间的相互作用。使用人工启动子系统评估CytR的不同结合方式以及CytR与CRP结合位点的不同排列方式的作用。这将允许对CytR操作员的结构和监管位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分析结果相结合，以建立差异基因调控的综合动力学模型。在相互作用或与RNAP相互作用中存在缺陷的CytR和CRP突变体将被用于评估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。本项目由生物科学理事会分子和细胞生物科学部的基因表达生物化学项目和分子生物物理学项目支持。

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.