Title
The dimerization site-2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation
Author
Yuki Yamanaka, Ricksen S Winardhi, Erika Yamauchi, So-ichiro Nishiyama, Yoshiyuki Sowa, Jie Yan, Ikuro Kawagishi, Akira Ishihama, Kaneyoshi Yamamoto
Year
2018
Journal
Journal of Biological Chemistry
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 site-1 and site-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 an 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 these four variants formed condensed complexes on DNA, whereas wild-type 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 the dimerization site-2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing.
Instrument
J-820
Keywords
Circular dichroism, Secondary structure, Biochemistry