Fig. 4: Characterization of Pol II dissociation from DNA in the post-termination state.
From: Single-molecule reconstruction of eukaryotic factor-dependent transcription termination
a Typical termination trajectories showing sequential dissociation of RNA (Cy3 disappearance, dash line) and Pol II (Cy5 disappearance) on bioPol II/RNA-Cy3/DNA-Cy5(+3) TECs. b Histograms of the time elapsed between RNA release and Pol II dissociation under the effect of 10 µg ml−1 heparin, i.e., Pol II dissociates before (gray), simultaneously with (navy), or after (purple) RNA release. Orange represents Pol II retaining on DNA over the course of the recording. The mean times (1/k4) of purple events are 36 ± 9 s (SEM, N = 74) and 2.1 ± 0.3 s (SEM, N = 120), respectively. d As for (b) but obtained on bioPol II/RNA-Cy3/DNA+19∼+28mis-Cy5(+3) TECs with mismatches at +19∼ + 28 yielding increased dissociation times of 89 ± 11 s (SEM, N = 54) and 72 ± 10 s (SEM, N = 26), respectively. Schematic of Pol II dissociation pathways during Sen1-dependent termination on bioPol II/RNA-Cy3/DNA-Cy5(+3) TECs (c) and under the effect of DNA mismatches (e). f Typical trajectories when labeling Cy5 at the downstream DNA end (bioPol II/RNA-Cy3/DNA-Cy5(3’) TECs, 49/172 events). g The mean lifetimes of Cy5 PIFE appearance (upper panel) and its fraction (lower panel) versus different distances of Cy5 fluorophore from the termination site (mean ± SEM, N = 112, 49, 56, 14, and 19 respectively). Data better fit to one-dimensional diffusion35 model yielding diffusion coefficient of (1.3 ± 0.3) × 10−4 µm2 s−1 (SEM) and the fraction of 0.26 ± 0.02 (SEM, a reduced chi-square of 1.2 for 1D model, dark blue line, compared to that of 7.1 for 3D model, light-blue dash line). h Corresponded histograms of Cy5 PIFE ratio give Gaussian peaks at 1.77 ± 0.04, 1.35 ± 0.02, 1.43 ± 0.02, 1.61 ± 0.04, 1.72 ± 0.08 (SEM), respectively. i Termination efficiencies (mean ± SEM) of engineered Pol II TECs with no mismatch (170/350), mismatch (166/406) in the absence of heparin, and no mismatch (372/812), mismatch (96/284) in the presence of 10 µg ml−1 heparin. Source data are provided as a Source Data file.
