H-NS binds with high affinity to the Tn10 transpososome and promotes transpososome stabilization.

H-NS is a bacterial DNA-binding protein that regulates gene expression and DNA transposition. In the case of Tn10, H-NS binds directly to the transposition machinery (i.e. the transpososome) to influence the outcome of the reaction. In the current work we evaluated the binding affinity of H-NS for two forms of the Tn10 transpososome, including the initial folded form and a pre-unfolded form. These two forms differ in that IHF is bound to the former but not the latter. IHF binding induces a bend (or fold) in the transposon end that facilitates transpososome formation. However, the continued presence of IHF in the transpososome inhibits intermolecular transposition events. We show that H-NS binds particularly strongly to the pre-unfolded transpososome with an apparent K(d) of approximately 0.3 nM. This represents the highest affinity interaction between H-NS and a binding partner documented to date. We also show that binding of H-NS to the transpososome stabilizes this structure and propose that both high-affinity binding and stabilization result from the combined interaction between H-NS and DNA and H-NS and transposase within the transpososome. Mechanistic implications for tight binding of H-NS to the transpososome and transpososome stabilization are considered.