Figure 3.

Example single-molecule FRET data of mononucleosomes. (A) FRET histogram constructed from many nucleosomes before (blue bars) and after (red bars) ACF-catalyzed remodeling. The three initial peaks (blue bars) arise from the three distinct donor dye labeling configurations (single proximal donor, single distal donor, proximal and distal donor present as described in the text). The black line represents a fit with three Gaussians. Upon remodeling with ACF, the FRET values are shifted toward very low values (red bars). (B) Donor fluorescence (green), acceptor fluorescence (red), and FRET (blue) traces depicting ACF-induced remodeling of an individual nucleosome with a single proximal donor fluorophore. The first intermediate FRET plateau is shaded in yellow. The duration of the first translocation pause is labeled as t_p,1, and begin and end time points are indicated for the first intermediate FRET plateau. (C) Initial FRET value (before remodeling) as a function of the exit linker DNA length (n bp). The black line depicts a linear fit to the data with a slope of −0.051 ± 0.002. The deviation from the expected nonlinear dependence is caused by the flexible linkers connecting the dyes to the nucleosome or the DNA. For this ruler, data from nucleosomes with a single Cy3 dye on the proximal H2A subunit are used. (D) FRET distribution of the pauses. (E) Distribution of t_p,1, the duration of the first translocation pause.