Figure 5. Full-length GAF multimer undergoes 1D diffusion during target search.

a, Schematic of construct design. Plasmids with or without the hsp70 promoter sequence were digested with HindIII and concatenated using T4 DNA ligase. Exposed ends were then biotinylated with adaptors, resulting in dual-end biotinylated DNA carrying repeating hsp70 promoters. b, Set-up of dual optical tweezers for confocal microscopy of stretched DNA tethered between two streptavidin-coated polystyrene beads. c-d, Representative kymographs show AF488-GAF-FL fluorescence signal (cyan) on DNA over time in the absence (c, vector only) or presence of hsp70 promoter (d, vector+hsp70). White arrowhead shows GAF-FL binding directly to target from 3D; black arrowheads show GAF-FL undergoing 1D search before finding target; white asterisk shows direct 3D dissociation. e, Compiled plots of position versus time for GAF-FL trajectories on vector only DNA. 100 traces were collected for each condition and arranged to start from zero time. f, Compiled plot for GAF-FL positions over time on vector+hsp70 DNA. g, Average mean squared displacement (MSD) over time lag of all collected traces for vector only (purple) and vector+hsp70 DNA (blue). Shaded area reports standard error of the mean (SEM). N=100. h, Pie charts categorizing GAF-FL traces by DNA binding at onset of movie (left), and targeting directly (3D binding) or indirectly (1D sliding) (right). i, A representative kymograph of GAF-ΔPOZ on vector+hsp70 DNA. j, 1-CDF comparing dwell times of GAF-ΔPOZ on vector+hsp70 DNA (red, τ = 1.67 s), GAF-FL on vector+hsp70 (blue, τ = 43.4 s) and GAF-FL on vector-only (purple, τ = 21.0 s). k, Single-trace diffusion coefficients for GAF-FL on vector+hsp70 and vector-only DNA and GAF-ΔPOZ on vector+hsp70 DNA. Error bars are SEM. Statistical differences were determined by unpaired t-test (n=100, **** = p < 0.0001).