title: H-NS binds with high affinity to the Tn10 transpososome and promotes transpososome stabilization.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Wardle
authors.forename: Amanda
authors.initials: A
authors.lastname: Chan
authors.forename: David B
authors.initials: DB
authors.lastname: Haniford
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000737:chemistry; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009690:Nucleic Acid Conformation
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria; D004247:DNA
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