DNA binding properties of integration host factor and effects on gene expression.

摘要

Escherichia coli are adept for survival in a wide range of environments, varying in the availability of nutrients, temperature, pH, osmotic stress, and even oxygen levels. In order to accomplish this feat, each cell must detect and respond to environmental stimuli promptly and in an efficient manner specific to its current condition. Oftentimes, changes in environmental settings lead to temporary shifts in the cellular energy levels, which in turn affect the activities of enzymes that utilize ATP. These changes in cellular energy levels that accompany environmental shifts are generally correlated with shifts in DNA supercoiling levels (van Workum et al., 1996). This indirect link between environmental stimuli and DNA supercoiling levels can act as a sensor of changing conditions. Most genes have an optimal supercoiling level for expression, and thus changes in DNA supercoiling may lead to global changes in basal levels of gene expression.;Previous work at the Hatfield laboratory has shown that the protein Integration Host Factor, IHF, influences gene regulation in a supercoiling-dependent manner. For example, the ilvGMEDA operon is regulated by an IHF protein-mediated translocation of superhelical energy from an upstream stress-induced DNA duplex destabilization (SIDD) site to a downstream promoter site (Parekh and Hatfield, 1996). This mechanism provides local superhelical energy to the promoter to amplify transcriptional activity, depending on the supercoiling levels of the cell. As DNA supercoiling varies in response to the environmental growth state of the cell, we predict variation in the basal level expression of genes regulated by this mechanism during environmental shifts. To test this hypothesis (1) we predicted IHF binding sites throughout the genome based on in vitro assays of a systematic set of de novo binding sites. (2) We determined gene expression profiles and measured the negative DNA supercoiling levels and energy charge of otherwise isogenic +/-IHF E. coli K12 strains during aerobic to anaerobic growth transitions. (3) We identified new genes potentially regulated by IHF and/or DNA supercoiling.