Fig. 4. ZntR_apo enhances facilitated unbinding of Zur_Zn from DNA in vitro.

a Histogram of E_FRET trajectories of immobilized 31-bp DNA^Cy3 interacting with ${\text{Zur}}_{\text{Zn},\text{D}49\text{A}}^{\text{Cy}5}$ (1 nM). Colored lines: Gaussian resolved fits, where each color corresponds to one ${\text{Zur}}_{\text{Zn},\text{D}49\text{A}}^{\text{Cy}5}$ dimer at one of the two dyads of Zur box in two orientations, as shown by the inset cartoons. Black line: overall fit. Bin size = 0.02. b Same as (a), but with ${\text{Zur}}_{\text{Zn},\text{D}49\text{A}}^{\text{Cy}5}$ at 4 nM. E₇ state (green-shaded) only appears when two ${\text{Zur}}_{\text{Zn},\text{D}49\text{A}}^{\text{Cy}5}$ dimers are bound to DNA. c Same as (a), but with ${\text{Zur}}_{\text{Zn}}^{\text{Cy}5}$ (4 nM). Cartoons show DNA-bound ${\text{Zur}}_{\text{Zn},}^{\text{Cy}5}$ with four different combinations of two dimer-bound form. d Same as (c) but with an introduction of unlabeled ZntR_apo (200 nM). E₃ state (blue-shaded) only appears when a single Zur_Zn dimer is bound to DNA. Histograms for (a–d) are compiled from 405, 433, 255, and 168 E_FRET trajectories, respectively. e Apparent unbinding rate constant k₋₁ of ${\text{Zur}}_{\text{Zn}}^{\text{Cy}5}$ on the 31-bp DNA vs. its own concentration at 0 (black), 100 (orange), 200 nM (green) of ZntR_apo. Lines: linear fits; the slope is the facilitated unbinding rate constant k_f for ${\text{Zur}}_{\text{Zn}}^{\text{Cy}5}$. k_f are extracted from total 3238 dwell times. f ZntR_apo-concentration-dependent k_f for ${\text{Zur}}_{\text{Zn}}^{\text{Cy}5}$ extracted from (e). k_f are extracted from 1005, 1339, 894 dwell times for 0, 100, 200 nM of ZntR_apo, respectively. Line: linear fit. g The binding rate constant k₁ for ${\text{Zur}}_{\text{Zn}}^{\text{Cy}5}$ to 31-bps DNA shows no significant dependence on [ZntR_apo]. k₁ are extracted from 249, 569, 493 dwell times for 0, 100, 200 nM of ZntR_apo, respectively. Line: horizontal line fit. The binding and unbinding rate constants are extracted from single exponential fits (y = A*exp(-k*τ)) of the distributions of dwell time (Supplementary Fig. 34, 35), and their error bars are 90% confidence intervals from fits. Source data are provided as a Source Data file.