Fig. 1

Experimental validation of Pol II initiation, elongation, and termination. a DNA containing an unpaired 12-base bubble flanked by Ec his terminators is tethered and torsionally constrained between a glass surface and a magnetic bead. b Time-trace obtained on negatively supercoiled Pol2-144-444-his construct shows the expected increases in DNA extension as small pulses, characterized by both their change in extension and their duration (Δl, Δt). Data were collected at 31.0802 Hz and all time traces were filtered at 0.5 Hz (red line). c 2D representation of transcription pulses reflecting their (Δl, Δt) coordinates (N = 407 events). d Lifetime projection of the 2D plot scaled in log is fit by a double-gaussian (red line, reduced chi-square is 0.75), yielding two peak durations: 10.0 ± 0.4 s (SEM) and 26.1 ± 1.6 s (SEM). Two sub-populations are selected based on the peak duration ± 0.7 SD: cyan, 10.0 ± 1.7 s (SD = 2.5 s for peak one); blue, 26.1 ± 10.1 s (SD = 14.5 s for peak two). Inset: linear scale of lifetime projection. The dashed violet line represents fitting to a single-gaussian with a reduced chi-square of 4.57. e Amplitude projection of the 2D plot sub-populations is fit to a single Gaussian (cyan line, giving a mean apparent unwinding of 9.9 ± 0.9 bp (SEM); blue line, 9.2 ± 0.3 bp (SEM); orange line; 8.3 ± 0.2 bp (SEM) in the blebbed bubble). f Dependence of the mean pulse lifetime on substrate length (blue) and inverse NTP concentration (red). Linear fitting of the length-dependence data (blue line) to the model t = L/v + t₀, where L is the transcript unit length, v the velocity, and t₀ the intercept gives v = 14.9 ± 0.4 bp/s and t₀ = 1.3 ± 0.6 s. Nucleotide-dependence data are fit to a Michaelis–Menten model (red line) yielding affinity for NTPs K_M = 230 ± 66 μM and V_max = 16.7 ± 2.6 bp/s. Error bars represent SEM; typically at least 200 events were collected for each NTP concentration. Source data are provided as a Source Data file