Dynamics of Promoter Occupancy by the Bacterial DNA-Binding Protein H-NS; Transcriptional Repression Viewed in the Milliseconds Time Scale

Histone-like Nucleoid Structuring protein (H-NS) is a well-characterized DNA-binding protein, known to condensate DNA and to act as a transcriptional repressor on bacterial Gram-negative genes. The proposed mechanism of repression is thought to occur via binding of the protein at multiple binding sites, followed by oligomerization of the protein and spreading to neighbor regions. These events induce a distortion of the double helix axis, leading to DNA curvature. As a consequence, the activity of RNA polymerase at the promoter sites is inhibited due to entrapping or to exclusion from the target sequence. We have investigated the interaction of H-NS with the promoter region of VirF, a gene responsible for the multistep pathogenicity cascade of Shigella flexneri. The results of footprinting experiments carried out at equilibrium in the presence of H-NS or its monomeric DNA binding domain (H-NSctd), indicate the presence of more than ten primary binding sites. These nucleation points have been indexed over a millisecond time scale by quantitative analyses of time-resolved hydroxyl radical footprinting experiments. Furthermore, fluorescent polarization studies carried out with six DNA fragments (22mer) corresponding to slow, medium and fast binding sites allowed us to determine the kinetic parameters of the interaction between H-NS and the isolated DNA targets. The combination of biomolecular and biophysical approaches will lead to a molecular model where the DNA-protein interactions, analysed on a temporal scale, provide a dynamic view of the mechanism of gene repression.