Upstream bubble collapse and transition into elongation are delayed in the P266L mutant.
(A) Real time 2AP fluorescence monitors upstream bubble collapse. Representative time courses of 2-AP fluorescence changes from position −4NT in individual walking experiments (+8, +9, +12, and +15) were observed in the stopped-flow setup. The initial increase indicates rapid opening of the promoter and the decrease indicates bubble collapse. (B) Single molecule FRET histograms measure the rate of promoter unbending by WT and P266L T7 RNAP at +9. The number of DNA molecules that were analyzed to draw the smFRET histograms are as follows: P266L: 3110 at 1 min; 4164 at 5 min; 4883 at 33 min. WT: 3444 at 1 min; 3432 at 5 min; 5855 at 34 min. The x-axis shows corrected FRET (equation 2) and the y-axis represents the frequency of transcription complexes with the respective FRET values. Low FRET is observed in the elongation complex (EC) and high FRET is observed in the initiation complexes (IC). An increase in the low FRET population is observed over time after stalling at +9 using GTP+ATP+CTP+3′dUTP. Concentration of T7 RNAP-DNA, GTP, and NTPs was 10 nM, 1 mM, and 500 µM, respectively. (C) The fraction of EC versus time was fit to a single exponential function. The error bars in Fig 5C is the standard error from fitting smFRET histogram to a single exponential function. The WT (red circles) transitions to EC faster than P266L (black circles) at position +9 (τ = 4.5±1 min for P266L and 1.7±0.3 min for WT). The cartoon shows the layout of the smFRET experiments.