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

Nucleic Acids Research
Ranjit GulvadyJie Yan

Abstract

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.

Citations

Jul 1, 2020·Frontiers in Microbiology·Karin BischofGünther Koraimann
Dec 13, 2019·PLoS Genetics·Subhash C VermaSankar L Adhya
Dec 22, 2020·Nucleic Acids Research·Xu LiZhongbo Yu
Jan 26, 2021·The Journal of Biological Chemistry·Lin LiangZhongbo Yu
Nov 5, 2019·Current Opinion in Chemical Biology·Xiaodan ZhaoJie Yan
Jul 19, 2019·Biochemistry·Yinan WangBenjamin T Goult

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BETA
footprinting
PCR

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