(A) (Top) Design of experiment monitoring promoter unwinding in real time. Grey, RNAP; orange, RNAP clamp; purple dot, RNAP active-centre; black, ds-DNA; blue, ss-DNA; light green, Cy3 on ds-DNA; dark green, Cy3 on ss-DNA. (Bottom) A cropped area (0.94 μm × 1.034 μm) of the field of view, showing appearance and enhancement of fluorescence signal from binding of single Cy3-labelled promoter fragment to an immobilised RNAP molecule. (B) (Left) Time trajectories of intensity from Cy3 on downstream segment of promoter bubble. Black, raw intensity; dark blue, idealised intensity; hidden Markov model (HMM)-assigned states: no promoter (black bars), closed promoter (light yellow bars) and open promoter (green bars). Frame rates: 50 ms, top and middle; 200 ms, bottom. Laser powers: 0.60 mW, top and middle; 0.15 mW, bottom. (Right) Dwell-time histograms of promoter state before unwinding, tUNWIND. (C) (Left) Time trajectories of intensity from Cy3 on upstream segment of promoter bubble. Colours as in B. Frame rates: 50 ms. Laser powers: 0.6 mW. (Right) Dwell-time histograms of promoter state before unwinding, tUNWIND. (D) Table comparing unwinding times for different promoter constructs.