Biophysical Studies of H-NS Binding to DNA

Abstract

Heat-stable nucleoid structuring protein (H-NS) is an abundant prokaryotic protein that organizes chromosomal DNA and plays an important role in gene silencing. Here we report that H-NS can have two distinct binding modes that can be switched from one to another by changes in divalent ion concentrations. Further, we show that the switching does not require dissociation of H-NS from the DNA. Our finding resolves an important controversy in the field in which mutually exclusive observations of H-NS/DNA interaction were reported. In single-molecule manipulation experiments, one binding mode leads to stiffening of DNA backbone, while the other leads to DNA folding. AFM imaging showed that the stiffening is caused by polymerization of H-NS along DNA starting from a few nucleation sites, while the folding is caused by formation of large DNA hairpins. The transition from stiffening to bridging occurs when the magnesium or calcium concentration is around or above 5 mM, suggesting that both binding modes are likely present at physiological conditions. Furthermore, the susceptibility of the two binding modes to physiological stimuli (pH and temperature), which are known modulators of H-NS activity, are discussed.