Fig. 7. T710 and H842 are critical regulators of translocation by Mfd.

a EM-based model of DNA-bound Mfd with boxed region highlighting the structural environment of T710 and H842 residues. The resolution of our reconstruction precludes precise determination of the conformation of motif IVa loop and of amino-acid rotameric forms. Crucial residues are indicated as Cα spheres, H842 in green, T710A in yellow and E1045, D1048, and R1049, mutated in Mfd D7^AAA in red. b–c TFO displacement assays carried out with Mfd^D7−AAA b and Mfd c variants. Percentage of remaining TFO-Triplex is plotted vs time to show translocation of Mfd variants. Plot shows means ± SD (n = 3), with error bars often smaller than symbols. d–e Electrophoretic separation of TFO displacement assays carried out with Mfd^D7−AAA. Representative gels for Mfd^D7−AAA d and Mfd e +/-ATP and +AMPPNP are shown. Example gels for Mfd, Mfd^{D7−AAA;T710A} and Mfd^D7AAA;H842A variants are shown in Supplementary Fig. 8 (n = 3). f Steady-state ATPase activities of Mfd variants normalized to wild-type Mfd activity. Data represent means ± SD (n ≥ 3). **denotes p < 0.01, ****p < 0.0001 and ns, p > 0.05 (unpaired two-tailed t test). The p value for Mfd^D7AAA;T710 with/without DNA was 0.0012. g, h Equilibrium dissociation constants for the Mfd-dsDNA interaction derived from fluorescence anisotropy binding curves obtained by titrating increasing amounts of protein into a mixture of 10 nm HEX-labeled 40mer DNA and 2.0 mm ATPγS e and ADP•AlF_x. Data represent mean values ± SD (n = 3). K_D values are tabulated in Supplementary Table 1. Source data are provided as a Source Data file.