Nucleic Acids Research Advance Access published online on August 20, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp672
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Molecular Biology |
H-NS binds with high affinity to the Tn10 transpososome and promotes transpososome stabilization
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1
*To whom correspondence should be addressed. Tel: +1 519 661 4013; Fax:+1 519 661 3175; Email: haniford{at}uwo.ca
Received June 25, 2009. Revised June 27, 2009. Accepted June 29, 2009.
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 Kd of 
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.