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

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