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 language | English (US) |
|---|---|
| Pages (from-to) | 10216-10224 |
| Number of pages | 9 |
| Journal | Nucleic acids research |
| Volume | 46 |
| Issue number | 19 |
| DOIs | |
| State | Published - Jan 1 2018 |
| Externally published | Yes |
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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 journal › Article
}
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.
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U2 - 10.1093/nar/gky826
DO - 10.1093/nar/gky826
M3 - Article
C2 - 30239908
AN - SCOPUS:85056285531
VL - 46
SP - 10216
EP - 10224
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 19
ER -