Fig. 3: Mfd moving through a bound dCas complex.
a, Flowchart of a single-molecule Mfd translocation assay. Some DNA tethers were used as controls to assay bound protein locations before ATP addition. Other tethers were used to assay bound protein locations after an ATP chase time of Δt = 8 min. During the ATP chase, Mfd was able to translocate along DNA, probably still interacting with a nontranscribing RNAP. DNA was always unzipped in the same direction as Mfd translocation. b, Representative traces of Mfd colliding with dCas9 and moving past dCas9, when approaching dCas9 from the PAM-distal side. Naked DNA traces are shown in black. c, Mfd move-through efficiency for Mfd encountering a bound dCas from either the PAM-distal side or the PAM-proximal side. Results from both dCas9 (top) and dCas12a (bottom) are shown. For each sample chamber, both control traces and noncontrol traces were taken to obtain the move-through efficiency for that chamber. Each type of experiment was repeated using n = 6 biologically independent sample chambers. Move-through values were calculated for each sample chamber (black dots), and the mean value and s.e.m. of these repeats are also shown. Source data containing traces for b and Mfd move-through values for c are provided.
