Dimerization site 2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation

Yuki Yamanaka,Ricksen S Winardhi,Erika Yamauchi,So-Ichiro Nishiyama,Yoshiyuki Sowa,Jie Yan,Ikuro Kawagishi,Akira Ishihama,Kaneyoshi Yamamoto

doi:10.1074/jbc.ra117.001425

Yuki Yamanaka, Ricksen S Winardhi + Show 7 more

Open Access

https://doi.org/10.1074/jbc.ra117.001425

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Abstract

The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing.

Highlights

We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing
Recent analysis of the linker identified a role for the five charged residues in initial engagement with DNA, 6 The abbreviations used are: nucleoid-associated proteins (NAPs), nucleoid-associated protein; GAD, glutamic acid– dependent acid resistance; AFM, atomic force microscopy; electrophoretic mobility assay (EMSA), electrophoretic mobility shift assay; LB, Luria–Bertani; IPTG, isopropyl 1-thio-␤-D-galactopyranoside
H-NS is composed of two discrete domains, an N-terminal oligomerization consisting of dimerization sites 1 and 2 and a C-terminal DNA-binding domain connected by a flexible linker

Summary

To whom correspondence should be addressed

We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing. Molecular analysis of truncated H-NS proteins led to predictions that E. coli H-NS (137-amino acid residues) consists of an N-terminal oligomerization domain (residues 1–79) [7] and a C-terminal DNA-binding domain (residues 95–137) [7, 25]. Most substitutions in the DNAbinding domain mapped within the core DNA-binding motif (TWTG-GR-P) between residues 108 –116 (Fig. 1, A and C). At least three substitutions in site 1 (R15E, L26P, and L30P) fail to form a rigid nucleoprotein filament [14, 31, 32], resulting in loss of gene silencing in vivo. Amino acid substitutions can disrupt this site-dependent domain– domain communication, abrogating gene silencing. The Cys-scanned H-NS series constructed in this study will provide a useful tool to analyze H-NS mutants by cysteine modification

Results

Discussion

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