Figure 2

H-NS binding assays. (A) H-NS and transposase were added simultaneously to the indicated ³²P-labeled transposon end substrate DNAs where indicated. Binding reactions were analyzed by gel electrophoresis on a native 5% polyacrylamide gel and subject to phosphorimager analysis. In each experiment transposase and substrate DNA concentrations were kept constant, while the concentration of H-NS was varied as indicated. Positions of transpososome (T'some), H-NS-shifted transpososome (H-NS-T'some) and unbound substrate DNA are indicated. In (A) lane 1 of the 'WT ME' gel was originally loaded in the last lane of the gel and was moved without alteration to the first lane of the gel. (B) Binding isotherms of fractional saturation as a function of H-NS concentration for H-NS binding to the wild-type ME and the OE transpososomes. Each binding isotherm was derived from four independent binding experiments similar to those depicted in (A). The percent transpososome complex shifted by H-NS was determined and these data were fit to a quadratic equation as described in Methods. The fits were used to provide estimates for the observed dissociation constant, obs K_d for the wild type mosaic end (WT ME) and the outside end (OE).