Role of Heteromeric Protein - Protein Interactions in Differential Gene Expression

9728186本研究的目的是了解E.ColiCytR调节子基因差异表达的组合机制(S)，该基因家族由九个独立的转录单位组成。转录单位受两种调节蛋白相互作用的协调调节：(1)环-AMP受体蛋白(CRP)，一种在细菌中广泛使用的转录激活蛋白；(2)CytR，一种调节子特异性抑制物，也是LacI同源抑制物家族的成员。该调控的一个关键特征是不同顺反子在激活、抑制和诱导的程度上各不相同。与真核生物中的情况类似，这种差异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，而更多的全局调控是由CRP介导的。大多数CytR调控的启动子的一个不寻常的特征是它们含有串联的CRP位点，据信这些位点通过不同的机制激活转录。与大多数细菌抑制物不同，CytR不是独立地抑制转录，而是通过与一系列DNA位点结合来调节CRP介导的激活，从而导致CytR和CRP之间的合作和竞争结合作用。在受CytR调控的启动子中，CytR位点的数量和排列有很大的不同，这似乎对它们的不同调控有很大贡献。本研究的目的是：1)了解CRP和CytR之间的协同和竞争相互作用在介导RNA聚合酶差异激活中的作用；2)确定CytR-CRP相互作用在协同DNA结合中的作用。为了实现第一个目标，我们将系统地比较CRP、CytR和两个受CytR调控的启动子之间的相互作用的能量学。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。为了实现后者，将通过分析AL超速离心法研究溶液相互作用和胞苷与CytR结合的影响。多个非连锁基因的协调调控是基因表达的基础。在转录水平上，这是由调控蛋白-DNA复合体的协作、特定部位组装控制的。一种常见的机制使用组合策略，既涉及相对常见的调节蛋白，也涉及特定于基因、组织或细胞类型或有机体发育阶段的蛋白质。通过与不同基因中常见的DNA位点的不同排列结合，相同的调节蛋白可以形成非常不同的蛋白质-DNA复合体。通过这种方式，即使在由相似的调控蛋白互补控制的情况下，基因也可以进行差异表达。此外，通过改变一个或几个因子的活性，可以很容易地控制整个基因表达程序。细胞色素R调节子是在分子水平上理解的一个特别重要的系统，因为它涉及真核和原核生物基因调控的许多基本问题，如协调基因调控，C反应蛋白介导的激活机制(S)，以及不同调控蛋白之间的相互作用如何控制蛋白质-DNA复合体的协同组装。

9728186 Senear The goal of this research is to understand the combinatorial mechanism(s) of differential gene expression in the E. coli CytR regulon, a gene family consisting of nine unlinked transcriptional units. The transcriptional units are regulated coordinately by the interplay of just two regulatory proteins: ( 1 ) cyclic-AMP receptor protein (CRP) a widely used transcriptional activator in bacteria; (2) CytR, a regulon-specific repressor and a member the LacI family of homologous repressors. A key feature of the regulation is that the different cistrons differ from one another in extents of activation, repression and induction. Similar to the situation in eukaryotes, this differential regulation is achieved by nesting levels of local repression, mediated by CytR on the more global regulation mediated by CRP. An unusual feature of most CytR regulated promoters is that they contain tandem CRP sites, which are believed to activate transcription via different mechanisms. Unlike most bacterial repressors, CytR does not repress transcription independently, but instead modulates CRP-mediated activation by binding to arrays of DNA sites that result in both cooperative and competitive binding interactions between CytR and CRP. The number and arrangement of CytR sites varies substantially among CytR regulated promoters and this appears to contribute significantly to their differential regulation. The objectives of the research are: 1) to understand the roles of cooperative and competitive interactions between CRP and CytR in mediating differential activation of RNA Polymerase; 2) to determine the role of CytR-CRP interactions in cooperative DNA binding. To accomplish the first, a systematic comparison of the energetics of interaction among CRP, CytR and two CytR-regulated promoters will be made. 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. To accompli sh the latter, solution interactions and the effect of cytidine binding to CytR thereon will be investigated by analytic al ultracentrifugation. Coordinate regulation of multiple, unlinked genes is fundamental to genetic expression. At the transcriptional level, this is controlled by cooperative, site-specific assembly of regulatory, protein-DNA complexes. A common mechanism uses a combinatorial strategy, involving both relatively common regulatory proteins and also proteins that are specific to either the gene, the tissue or cell type, or the development stage of the organism. By binding to variable arrays of DNA site, as are generally found in different genes, the same regulatory proteins can form very different protein-DNA complexes. In this manner, genes can be differentially expressed even while controlled by a similar complement of regulatory proteins. In addition, whole programs of gene expression are easily controlled by changing the activity of one or a few factors. The CytR regulon is a particularly important system to understand at a molecular level because it addresses many basic issues of both eukaryotic and prokaryotic gene regulation, such as coordinate gene regulation, the mechanism(s) of CRP-mediated activation, and how interactions among different regulatory proteins control the cooperative assembly of protein-DNA complexes.