Fig. 2: Characterization of the formation and resolution of the Sen1-Pol II TEC intermediate.
From: Single-molecule reconstruction of eukaryotic factor-dependent transcription termination
a Typical trajectories showing Pol II termination mediated by Sen1 HD-SNAP649 (biotinylation on Pol II and on DNA in the upper and lower panels, respectively). Three zones are identified: a green zone, representing Pol II TEC initiating transcription and then stalling (i.e., the pre-termination state); a pink zone, representing the formation of a Sen1-Pol II TEC intermediate (i.e., the termination state) as reflected by the appearance of Sen1 HD-SNAP649 fluorescence in the red channel and its FRET-interactions with RNA-Cy3; and a yellow zone, representing a state after resolution of Sen1-Pol II TEC intermediate (i.e., the post-termination state). Simultaneous dissociation of Sen1 HD-SNAP649 and RNA-Cy3 are observed. b Histogram showing the number of events versus the lag time, in units of video frames (50 Hz acquisition frequency), between the disappearance of Sen1 HD-SNAP649 fluorescence and the disappearance of Cy3 fluorescence. Zero represents the simultaneous disappearance of Sen1 HD-SNAP649 and RNA-Cy3 (N = 229). Events which show a non-zero lag between disappearance events follow a single exponential decay giving an average of 1.8 ± 0.9 frames (SEM, N = 14) and a predicted number of 11.8 ± 6.5 (SEM) events at 0 frame. c Duration histograms of the Sen1-Pol II TEC intermediate at various ATP concentrations are fit to single-molecule Michaelis–Menten function (see “Methods” for details), giving k1 = 2.20 ± 0.32 (SEM, N = 143), 2.54 ± 0.40 (SEM, N = 179), 5.12 ± 0.98 (SEM, N = 204), and 5.87 ± 1.19 s−1 (SEM, N = 229) for 20 µM, 40 µM, 200 µM, and 1.1 mM ATP concentrations, respectively, and k2 = 8.69 ± 2.03 s−1 (SEM) with a reduced Chi-square of 1.8. A mean lifetime of 0.25 ± 0.02 s (SEM) is calculated for the 1.1 mM ATP condition. d The values for k1 are further fit to a classical Michaelis–Menten model giving k1max = 5.9 ± 1.0 s−1 (SEM), and Km = 40 ± 15 µM (SEM, N = 143, 179, 204, and 229, respectively). Source data are provided as a Source Data file.
