Fig. 4

Stalled Pol II is fully dissociated from DNA upon action of Sen1 FL or Sen1 HD. a Schematic of the assay. Biotinylated Pol II is stalled on DNA, tethered to a magnetic bead, and placed in the magnetic trap. Transcription against a 1 pN force is restarted by addition of TFIIS and NTPs (24 out of 58 molecules resumed transcription). b Time-trace showing processive transcription by Pol II with interruptions due to long-lived pauses. Pauses shorter than ~10 s cannot be detected despite data filtering on the 2 s timescale. Detectable pauses are distributed according to single-exponential statistics with a mean of 29 ± 5 s (SEM, n = 67; left inset). When pauses longer than 10 s are removed, the velocity distribution can be fit to a Gaussian (red line) with mean velocity of 8.1 ± 0.6 bp/s (SEM, n = 43; right inset). c Sketch of the assay in the presence of Sen1 FL or Sen1 HD. d Time-trace showing release of stalled Pol II by 500 pM Sen1 FL (cyan; 76 Pol II molecules released out of 121), 500 pM Sen1 HD (blue; 52 Pol II molecules released out of 111). In controls carried out without Sen1 only 35 of 127 Pol II molecules released spontaneously. Inset shows the distribution of time elapsed between addition of translocase and release of Pol II. By fitting to a cumulative expression of single exponential function: f(t) = A*(1–exp(−t /t₀)), where A is the fraction of molecules which ultimately dissociate, t₀ is the time constant of this dissociation, we obtained the mean time for releasing stalled Pol II as 2935 ± 1840 s (SEM, n = 35) for no Sen1, 265 ± 69 s (SEM, n = 77) for Sen1 FL, and 963 ± 278 s (SEM, n = 52) for Sen1 HD. The final fraction of dissociation is 0.37 ± 0.15 for no Sen1, 0.59 ± 0.03 for Sen1 FL, and 0.45 ± 0.05 for Sen1 HD. Source data are provided as a Source Data file