Global Regulatory Networks in Escherichia Coli

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DESCRIPTION (provided by applicant): We have previously described DNA supercoiling-dependent mechanisms involving protein (IHF or FIS)- mediated translocation of local superhelical energy from one supercoiling-induced duplex destabilized (SIDD) region on the chromosome to another that serve to coordinate the basal levels of expression of the itvGMEDA, leuV, and ilvYC operons of the ilv regulon of Escherichia coli, both with one another and with the nutritional and environmental states of the cell. Here we propose to employ computational and biological methods to determine the extent to which these mechanisms are used for the global regulation of gene expression in this model organism. More specifically, we will use methods involving computational prediction and experimental verification to bring together three separate lines of inquiry to determine all the genes of E. coli that have the catenation of properties needed for IHF-mediated regulation by a mechanism involving a binding-induced transmission of destabilization. First, we will use computational methods to predict the locations of all the SIDD sites on the E. coli chromosome at superhelical densities encountered in otherwise isogenic wild-type, topoisomerase (topA, or gyrB) deficient E. coli strains. Second, we will develop and apply a novel method to computationally search the E. coli genome to identify all high affinity IHF binding sites. Third, we will use DNA microarrays to obtain gene expression profiles in IHF + and IHF- cells at the superhelical densities encountered in these strains. These data will allow us to identify genes (operons) that are regulated either by DNA supercoiling, or by IHF, or both. Genes whose upstream flanks contain a SIDD site that coincides with or overlaps a strong IHF binding site, and which are shown to have IHF dependent expression will be identified, and subjected to further experimental study to verify: l) that superhelicity destabilizes the predicted SIDD site; 2) that IHF binds at the predicted location; 3) that changes in duplex destabilization patterns alone, without changes in the local base sequence, affect gene regulation; and 4) that IHF binding regulates this expression in a DNA-supercoiling-dependent manner in an in vitro system.