Combinatorial Regulation of Differential Gene Expression in E. coli

University Of California-Irvine, Irvine CA

Investigators

Abstract

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.