Fig. 3: Characterization of Sen1-SNAP649 action on Pol II TEC tethered via a biotinylated RNA.

a Typical trajectories showing transcription termination of Pol II/bioRNA/DNA_58bp-Cy3 TECs by Sen1 HD-SNAP649. A post-termination state showing persistent interaction of Sen1 HD-SNAP649 with RNA as reflected by a lag time between disappearance of the DNA_58bp-Cy3 signal (purple zone) compared to those in Fig. 2a. b Lifetime distributions of the Sen1-Pol II TEC intermediate (pink zone) are globally fit to single-molecule Michaelis–Menten function (see “Methods”) giving k₁ = 0.84 ± 0.19, 3.21 ± 0.68, 4.59 ± 1.15, and 5.60 ± 1.61 s⁻¹ (SEM, N = 116, 160, 190, and 197 for 20 µM, 40 µM, 200 µM, and 2 mM ATP concentrations, respectively) and k₂ = 6.71 ± 2.01 s⁻¹ (SEM) with a reduced Chi-square of 1.8. c k₁ values are fit to classical Michaelis–Menten model giving k₁^max = 6.5 ± 1.6 s⁻¹ (SEM), and K_m = 109 ± 41 µM (SEM, N = 116, 160, 190, and 197, respectively). d k₁ values (mean ± SEM, N = 170, 195, 116, and 118, respectively) obtained using various RNA lengths at 20 µM ATP (see Supplementary Fig. 8a) are plotted versus the length of RNA that extends from the RNA exit channel of Pol II. e Lifetime distributions of Sen1 HD-SNAP649 residence in the post-termination state (purple zone) as a function of ATP concentration are individually fit to a single exponential function, giving a rate constant (k₃) of 1.99 ± 0.30, 4.21 ± 0.50, 4.25 ± 0.45, and 5.46 ± 0.48 s⁻¹ (SEM). f The rate constants from the prior panel follow a classical Michaelis–Menten model with k₃^max = 5.33 ± 0.40 s⁻¹ (SEM), and K_m = 27 ± 7 µM (SEM, N = 116, 160, 190, and 197, respectively). g Dissociation rate constant (mean ± SEM, N = 170, 195, 116, and 118) of Sen1 HD-SNAP649 in the post-termination state versus the length of RNA that extends from the RNA exit channel of Pol II (see Supplementary Fig. 8b). Source data are provided as a Source Data file.