H-NS cooperative binding to high-affinity sites in a regulatory element results in transcriptional silencing

Abstract

H-NS is a protein of the bacterial nucleoid involved in DNA compaction and transcription regulation. In vivo, H-NS selectively silences specific genes of the bacterial chromosome. However, many studies have concluded that H-NS binds sequence-independently to DNA, leaving the molecular basis for its selectivity unexplained. We show that the negative regulatory element (NRE) of the supercoiling-sensitive Escherichia coli proU gene contains two identical high-affinity binding sites for H-NS. Cooperative binding of H-NS is abrogated by changes in DNA superhelical density and temperature. We further demonstrate that the high-affinity sites nucleate cooperative binding and establish a nucleoprotein structure required for silencing. Mutations in these sites result in loss of repression by H-NS. In this model, silencing at proU, and by inference at other genes directly regulated by H-NS, is tightly controlled by the cooperativity between bound H-NS molecules.

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Figure 1: Schematic representation of the proU promoter.
Figure 2: DNase I footprints and quantification of H-NS binding on the proU promoter.
Figure 3: DNase I footprinting and KMnO4 reactivity of H-NS binding on the U162 and S162 fragments.
Figure 4: DNA stability plots.
Figure 5: Relative expression from the various proU constructs measured by fluorescence analysis.

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Acknowledgements

This work was supported by the Centre National de la Recherche Scientifique and the Agence Nationale pour la Recherche (projet MASTRIT). E.B. was funded by the Fondation pour La Recherche Médicale and the Ministère délégué à l'Enseignement supérieur et à la Recherche. We thank B. Robert and J. Gowrishankar for comments on the manuscript, I. Pemberton for his advice with the various constructions, G. Mitchison (University of Cambridge) for writing the DNA stability plot program and P. Bertin for kindly providing the strains.

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Correspondence to Sylvie Rimsky.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Footprinting on a modified proU sequence at positions +130 and +25. (PDF 174 kb)

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Bouffartigues, E., Buckle, M., Badaut, C. et al. H-NS cooperative binding to high-affinity sites in a regulatory element results in transcriptional silencing. Nat Struct Mol Biol 14, 441–448 (2007). https://doi.org/10.1038/nsmb1233

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