DNA Binding Is Not Sufficient for H-NS-mediated Repression ofproU Expression

Abstract

H-NS is a major component of bacterial chromatin and influences the expression of many genes. H-NS has beenshown to exhibit a binding preference for certain ATrich curved DNA elements in vitro. In this study we have addressed the factors that determine the specificity of H-NS action in vitro and in vivo. In bandshift studies, H-NS showed a slight binding preference for all curved sequences tested whether GC-based or AT-based; the specific architecture of the curve also influenced H-NS binding. In filter retention assays little difference in affinity could be detected for any sequence tested, including the downstream regulatory element (DRE) a downstream curved DNA element required for H-NS to repress transcription of the Salmonella typhimurium proU operon in vivo. A K d of 1-2 M was estimated for binding of H-NS to each of these sequences. In vivo, the distance between the proU promoter and the DRE, their relative orientations on the face of the DNA helix, and translation of the DRE had no major effect on proU regulation. None of the synthetic curved sequences tested could functionally replace the DRE in vivo. These data show that differential binding to curved DNA cannot account for the specificity of H-NS action in vivo. Furthermore, binding of H-NS to DNA per se is insufficient to repress the proU promoter. Thus, the DRE does not simply act as an H-NS binding site but must have a more specific role in mediating H-NS regulation of proU transcription.

The Cyclic AMP-Cyclic AMP Receptor Protein Complex Regulates Activity of the traJ Promoter of the Escherichia coli Conjugative Plasmid pRK100

“…Mobility shift assays with agarose gels were performed as described previously (16), except that the reaction volume was 20 l and the preparations were incubated for 15 min at 37°C. A 100-bp DNA ladder (MBI Fermentas) was used as the competitor DNA.…”

Roles of the DNA binding proteins H-NS and StpA in homologous recombination and repair of bleomycin-induced damage in Escherichia coli

“…On the other hand, there is another possibility to explain the deficiency of homologous recombination in the hns single or hns stpA double mutant. It is known that H-NS binds preferentially to highly curved DNA (Jordi et al, 1997) and can even bend non-curved DNA through its binding. Oligomerization domain of this protein seems to play a critical role in the bending (Spurio et al, 1997).…”

Structural Basis for H-NS-mediated Trapping of RNA Polymerase in the Open Initiation Complex at the rrnB P1

“…It has been shown in vitro that indeed H-NS binds with a higher affinity to strongly curved DNA when compared with noncurved or moderately curved DNA, independent of the base composition (29,30). Footprints of H-NS on the most strongly curved DNA fragment examined show specific protection of the curved region (31).…”