Fig. 3. SmFRET measurements show dynamic Zur_Zn-DNA interactions in vitro.

a Top: the DNA oligomer sequence from E. coli znuCB promoter and Cy3/biotin label positions for smFRET measurements. Pink shade: Zur box; pink arrows: two dyads for binding two Zur_Zn dimers; blue arrows: potential ZntR recognition dyad (Fig. 1b). Bottom: Two different-length DNA constructs. A single Cy5 is labeled at C113 or C158 on Zur. Zoom-in box: C113 and approximate C158 positions (shown on different monomers for clarity) in the dimeric Zur_Zn’s crystal structure (PDB: 4MTD⁴⁴; residues 153-171 are unresolved). b Representative single-molecule E_FRET trajectory of an immobilized 22-bp truncated DNA^Cy3 interacting with ${\text{Zur}}_{\text{Zn},\text{D}49\text{A}}^{\text{Cy}5}$ (Cy5 at C113) (4 nM). Pink line: raw data; red line: after non-linear filtering; blue line: mean value of each E_FRET state. Three E_FRET states (E₀, E₁, E₂) are denoted. Dwell times on E₀ state are designated as τ_unbound and those on higher states (E₁ and E₂) as τ_bound. c Two-dimensional histogram of the lower vs. higher E_FRET state values from single-molecule E_FRET trajectories, as in (b). Top/right: corresponding one-dimensional projections; red (black) lines: Gaussian resolved (overall) fits. d Histogram of E_FRET trajectories, as in (b). e Same as (d), but with ${\text{Zur}}_{\text{Zn}}^{\text{Cy}5}$ (Cy5 at C113) (4 nM). f Same as (d), but with ${\text{Zur}}_{\text{Zn}}^{\text{Cy}5-\text{C}158}$ (4 nM). Cartoons in (d–f) show free DNA and DNA-bound Zur_Zn in two binding orientations differentiated by the Cy5-label. The FRET donor (green sphere) and acceptor (red sphere) are drawn on DNA and Zur at their approximate locations. Histograms for (d–f) are compiled from 305, 389, and 238 E_FRET trajectories, respectively; bin size = 0.05. Source data are provided as a Source Data file.