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

Ranjit Gulvady, Yunfeng Gao, Linda Kenney, Jie Yan

Research output: Contribution to journalArticle

4 Citations (Scopus)

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 (K D ) 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 K D ) 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
Volume46
Issue number19
DOIs
StatePublished - Jan 1 2018
Externally publishedYes

Fingerprint

DNA-Binding Proteins
Hot Temperature
DNA
Proteins
Gene Silencing
Polymerization
Binding Sites
Prokaryotic Cells
Genomic Islands
Static Electricity
Protein Binding
Genome

ASJC Scopus subject areas

  • Genetics

Cite this

A single molecule analysis of H-NS uncouples DNA binding affinity from DNA specificity. / Gulvady, Ranjit; Gao, Yunfeng; Kenney, Linda; Yan, Jie.

In: Nucleic acids research, Vol. 46, No. 19, 01.01.2018, p. 10216-10224.

Research output: Contribution to journalArticle

Gulvady, Ranjit ; Gao, Yunfeng ; Kenney, Linda ; Yan, Jie. / A single molecule analysis of H-NS uncouples DNA binding affinity from DNA specificity. In: Nucleic acids research. 2018 ; Vol. 46, No. 19. pp. 10216-10224.
@article{b96706327a964613aabdbc11ad1d5df4,
title = "A single molecule analysis of H-NS uncouples DNA binding affinity from DNA specificity",
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 (K D ) 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 K D ) 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.",
author = "Ranjit Gulvady and Yunfeng Gao and Linda Kenney and Jie Yan",
year = "2018",
month = "1",
day = "1",
doi = "10.1093/nar/gky826",
language = "English (US)",
volume = "46",
pages = "10216--10224",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "19",

}

TY - JOUR

T1 - A single molecule analysis of H-NS uncouples DNA binding affinity from DNA specificity

AU - Gulvady, Ranjit

AU - Gao, Yunfeng

AU - Kenney, Linda

AU - Yan, Jie

PY - 2018/1/1

Y1 - 2018/1/1

N2 - 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 (K D ) 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 K D ) 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.

AB - 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 (K D ) 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 K D ) 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.

UR - http://www.scopus.com/inward/record.url?scp=85056285531&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85056285531&partnerID=8YFLogxK

U2 - 10.1093/nar/gky826

DO - 10.1093/nar/gky826

M3 - Article

VL - 46

SP - 10216

EP - 10224

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 19

ER -