Fig. 1: Single-molecule fluorescence characterization of Sen1-mediated Pol II transcription termination.

a Schematic of Pol II transcription initiated in the presence of ATP, UTP, and CTP, stalling, and extrinsic termination mediated by Sen1. b Typical trajectories showing (i) Pol II transcription elongation (light-gray zone) as reflected by the changes in Cy3/Cy5 fluorescence and in the corresponding FRET signal (∼0.6 → ∼0.8 → ∼0.2, blue, for FRET and ∼0.5 → ∼0.7 → ∼0.2, light blue, for corrected FRET’) in the upper two panels (green laser excitation) and Cy5 PIFE in the lower panel (red laser excitation), followed by (ii) Pol II stalling at the G-stretch (green zone) as reflected by stable Cy3/Cy5 fluorescence and FRET until (iii) Sen1 HD-mediated transcription termination as reflected by the abrupt disappearance of Cy3 fluorescence (vertical dash line). Simultaneous dissociation of DNA-Cy5 is shown in the lower panel. c Duration histograms of Pol II elongation (light-gray zone) are each fit to a single-Gaussian function, yielding peaks at 2.9 ± 0.2 s (SEM, N = 328), 2.7 ± 0.3 s (SEM, N = 412), 2.8 ± 0.2 s (SEM, N = 288) and 2.7 ± 0.1 s (SEM, N = 498) for 0, 3.5, 5, and 10 nM Sen1 HD, respectively. The fraction of Pol II restarting elongation is 328 out of 668 in the absence of Sen1 HD. d Histograms of Cy5 PIFE ratio calculated as the mean Cy5 intensity in the light-gray region over that in the green region, yielding peaks at 1.98 ± 0.03, 1.75 ± 0.02, 1.75 ± 0.02, 1.83 ± 0.02 (SEM) for 0, 3.5, 5, and 10 nM Sen1 HD, respectively. e Duration histograms of a stalled Pol II remodeled by Sen1 HD (green zone) are globally fit to single-molecule Michaelis–Menten function with photobleaching correction (see “Methods” for details), yielding a binding rate constant of k₊ = (6.8 ± 0.4) × 10⁶M⁻¹ s⁻¹ and k_- ≈ 0 with a reduced Chi-square of 1.0. N = 337, 417, and 564 events were collected for Sen1 concentrations of 3.5, 5, and 10 nM, respectively. Source data are provided as a Source Data file.