Mechanical conversion of low-affinity Integration Host Factor binding sites into high-affinity sites

Details Mechanical conversion of low-affinity Integration Host Factor binding sites into high-affinity sites Mechanical conversion of low-affinity Integration Host Factor binding sites into high-affinity sites

2009-04-13

arxiv.org/abs/0904.1900v1

Biology

Quantitative Biology

Biomolecules

Scientific paper

Although DNA is often bent in vivo, it is unclear how DNA-bending forces modulate DNA-protein binding affinity. Here, we report how a range of DNA-bending forces modulates the binding of the Integration Host Factor (IHF) protein to various DNAs. Using solution fluorimetry and electrophoretic mobility shift assays, we measured the affinity of IHF for DNAs with different bending forces and sequence mutations. Bending force was adjusted by varying the fraction of double-stranded DNA in a circular substrate, or by changing the overall size of the circle (1). DNA constructs contained a pair of Forster Resonance Energy Transfer dyes that served as probes for affinity assays, and read out bending forces measured by optical force sensors (2). Small bending forces significantly increased binding affinity; this effect saturated beyond ~3 pN. Surprisingly, when DNA sequences that bound IHF only weakly were mechanically bent by circularization, they bound IHF more tightly than the linear "high-affinity" binding sequence. These findings demonstrate that small bending forces can greatly augment binding at sites that deviate from a protein's consensus binding sequence. Since cellular DNA is subject to mechanical deformation and condensation, affinities of architectural proteins determined in vitro using short linear DNAs may not reflect in vivo affinities.

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