Role of Heteromeric Protein - Protein Interactions in Differential Gene Expression

9728186 Senear本研究的目的是了解大肠杆菌中差异基因表达的组合机制。coli CytR调节子是由9个不相连的转录单位组成的基因家族。转录单位仅通过两种调节蛋白的相互作用协调调节：（1）环AMP受体蛋白（CRP），其是细菌中广泛使用的转录激活剂;（2）CytR，其是调节子特异性阻遏物，并且是同源阻遏物的LacI家族的成员。调节的一个关键特征是不同的顺反子在激活、抑制和诱导的程度上彼此不同。与真核生物中的情况类似，这种差异性调节是通过局部抑制的嵌套水平来实现的，由CytR介导的对CRP介导的更全面的调节。大多数CytR调控启动子的一个不寻常的特征是它们含有串联CRP位点，据信这些位点通过不同的机制激活转录。与大多数细菌阻遏物不同，CytR不独立地抑制转录，而是通过结合DNA位点阵列来调节CRP介导的激活，从而导致CytR和CRP之间的合作和竞争性结合相互作用。CytR位点的数量和排列在CytR调节的启动子之间变化很大，这似乎对它们的差异调节有显着贡献。本研究的目的是：1）了解CRP和CytR之间的合作和竞争相互作用在介导RNA聚合酶的差异激活中的作用; 2）确定CytR-CRP相互作用在合作DNA结合中的作用。为了完成第一个，CRP，CytR和两个CytR调节的启动子之间的相互作用的能量学的系统比较。这些数据将结合转录起始的稳态和预稳态测定的结果，以开发差异基因调控的综合动力学模型。为了完成后者，将通过分析超离心研究溶液相互作用和胞苷与CytR结合的效果。 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，这是由调节性蛋白质-DNA复合物的协同、位点特异性组装控制的。一种常见的机制使用组合策略，涉及相对常见的调节蛋白以及对基因、组织或细胞类型或生物体的发育阶段具有特异性的蛋白。通过结合到DNA位点的可变阵列，如通常在不同基因中发现的，相同的调节蛋白可以形成非常不同的蛋白质-DNA复合物。以这种方式，基因可以差异表达，即使是在由调节蛋白的类似互补控制的情况下。此外，基因表达的整个程序很容易通过改变一个或几个因子的活性来控制。CytR调节子是在分子水平上理解的特别重要的系统，因为它解决了真核和原核基因调控的许多基本问题，例如协调基因调控、CRP介导的激活机制以及不同调控蛋白之间的相互作用如何控制蛋白质-DNA复合物的协同组装。

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.