The Role of LRP and H-NS in Transcription Regulation: Involvement of Synergism, Allostery and Macromolecular Crowding

LRP has recently been shown to interact with the regulatory regions of bacterial ribosomal RNA promoters. Here we study details of the LRP–rDNA interaction by gel retardation and high-resolution footprinting techniques. We show that a second regulator for rRNA transcription, H-NS, facilitates the formation of a higher-order LRP–nucleoprotein complex, probably acting transiently as a DNA chaperone. The macromolecular crowding substance ectoine stabilizes the formation of this dynamic complex, while the amino acid leucine, as a metabolic effector, has the opposite effect. DNase I and hydroxyl radical footprint experiments with LRP–DNA complexes reveal a periodic change of the target DNA structure, which implies extensive DNA wrapping reaching into the promoter core region. We show furthermore that LRP binding is able to constrain supercoils, providing a link between DNA topology and regulation. The results support the conclusion that the bacterial DNA-binding protein LRP, assisted by H-NS, forms a repressive nucleoprotein structure involved in regulation of rRNA transcription. The formation of this regulatory structure appears to be directly affected by environmental changes.