Role of Heteromeric Protein - Protein Interactions in Differential Gene Expression

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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.