H-NS mediated repression of the Escherichia coli bgl and proU operons

The histone-like nucleoid structuring protein H-NS is important in the organization of the bacterial chromosome and in global gene regulation in response to environmental stimuli and stress conditions. In Enterobacteriaceae such as Escherichia coli H-NS represses ~5 percent of all genes. Repression by H-NS is presumably mediated by binding of H-NS next to a promoter, and the formation of extended nucleoprotein complex, which inhibits transcription initiation. Although the specificity of binding of H-NS to DNA is low (it binds weakly specific to AT-rich curved DNA), some loci are very specifically repressed by H-NS including the E. coli bgl and proU operons. In both of these systems, upstream and downstream regulatory elements are required for efficient repression. In bgl H-NS binds 600 to 700 bp downstream to the promoter and in proU it binds 150 to 300 bp downstream. The analysis done here suggests that repression of proU and bgl by binding of H-NS to upstream and downstream regulatory elements is cooperative. Furthermore, it was shown that in the absence of the upstream regulatory element (URE), repression by H-NS binding to the downstream regulatory element (DRE) depends on the transcription rate. Termination factor Rho and co-transcriptional translation, which both modulate the transcription rate, were shown to also affect repression by H-NS via the DRE. Further experiments excluded, that H-NS acts as a roadblock to the transcribing RNA polymerase. In the bgl operon H-NS represses transcription elongation merely 2-fold and in proU it has no effect on elongation. These experiments include CAA-footprinting of stalled RNA polymerase transcription elongation complexes, Northern analysis, and a dual reporter gene system with the bgl and proU DRE, respectively, inserted in between uidA (?-glucuronidase) and lacZ (?galactosidase). In addition, the analysis of structural components in bgl revealed an intrinsic transcription pause site located in between the promoter and the bgl-DRE. However, the deletion of the pause did not affect repression. Additional deletion analyses suggest that the DNA segment upstream of the bgl-DRE is important for repression. The data shown here and ongoing experiments suggest that binding of H-NS to the DRE inhibits transcription initiation at the bgl and proU promoter, respectively. Possibly, H-NS bound to the DRE traps a DNA segment located upstream of the promoter resulting in DNA looping and repression of transcription initiation. Furthermore, the present work highlights the significance of the transcription rate and the process of transcription elongation in the modulation of H-NS mediated repression. Presumably, an increase in the transcription rate de-stabilizes the repressing complex formed by H-NS and thus causes full expression