A single molecule analysis of H-NS uncouples DNA binding affinity from DNA specificity

Ranjit Gulvady, Yunfeng Gao, Linda J. Kenney, Jie Yan

Research output: Contribution to journalArticlepeer-review

15 Scopus citations


Heat-stable nucleoid structuring protein (H-NS) plays a crucial role in gene silencing within prokaryotic cells and is important in pathogenesis. It was reported that H-NS silences nearly 5% of the genome, yet the molecular mechanism of silencing is not well understood. Here, we employed a highly-sensitive single-molecule counting approach, and measured the dissociation constant (KD) of H-NS binding to single DNA binding sites. Charged residues in the linker domain of H-NS provided the most significant contribution to DNA binding affinity. Although H-NS was reported to prefer A/T-rich DNA (a feature of pathogenicity islands) over G/C-rich DNA, the dissociation constants obtained from such sites were nearly identical. Using a hairpin unzipping assay, we were able to uncouple non-specific DNA binding steps from nucleation site binding and subsequent polymerization. We propose a model in which H-NS initially engages with non-specific DNA via reasonably high affinity (∼60 nM KD) electrostatic interactions with basic residues in the linker domain. This initial contact enables H-NS to search along the DNA for specific nucleation sites that drive subsequent polymerization and gene silencing.

Original languageEnglish (US)
Pages (from-to)10216-10224
Number of pages9
JournalNucleic acids research
Issue number19
StatePublished - Nov 2 2018
Externally publishedYes

ASJC Scopus subject areas

  • Genetics


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