Dataset Information

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.

SUBMITTER: Wardle SJ

PROVIDER: S-EPMC2764420 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Wardle Simon J SJ Chan Amanda A Haniford David B DB

Nucleic acids research 20090820 18

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 ...[more]

PMID: 19696075

Similar Datasets

Project description:BACKGROUND:The H-NS protein is a global regulator of gene expression in bacteria and can also bind transposition complexes (transpososomes). In Tn5 transposition H-NS promotes transpososome assembly in vitro and disruption of the hns gene causes a modest decrease in Tn5 transposition (three- to five-fold). This is consistent with H-NS acting as a positive regulator of Tn5 transposition. Molecular determinants for H-NS binding to the Tn5 transpososome have not been determined, nor has the strength of the interaction been established. There is also uncertainty as to whether H-NS regulates Tn5 transposition in vivo through an interaction with the transposition machinery as disruption of the hns gene has pleiotropic effects on Escherichia coli, the organism used in this study. RESULTS:In the current work we have further examined determinants for H-NS binding to the Tn5 transpososome through both mutational studies on Tn5 termini (or 'transposon ends') and protein-protein cross-linking analysis. We identify mutations in two different segments of the transposon ends that abrogate H-NS binding and characterize the affinity of H-NS for wild type transposon ends in the context of the transpososome. We also show that H-NS forms cross-links with the Tn5 transposase protein specifically in the transpososome, an observation consistent with the two proteins occupying overlapping binding sites in the transposon ends. Finally, we make use of the end mutations to test the idea that H-NS exerts its impact on Tn5 transposition in vivo by binding directly to the transpososome. Consistent with this possibility, we show that two different end mutations reduce the sensitivity of the Tn5 system to H-NS regulation. CONCLUSIONS:H-NS typically regulates cellular functions through its potent transcriptional repressor function. Work presented here provides support for an alternative mechanism of H-NS-based regulation, and adds to our understanding of how bacterial transposition can be regulated.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure