Fig. 1
Initial transcription monitored at the single-molecule level. a Representation of the premelted (turquoise font) WT DNA promoter used in the single-molecule experiments (Supplementary Fig. 1a). The – 35 and – 10 elements are represented in red. The promoter was donor labeled at – 15 position (green sphere) of the non-template DNA strand and acceptor labeled in + 15 (red sphere) position of the template DNA strand. An arrow above the base in orange font indicates the + 1 position. All the promoters used in the study are described in Supplementary Fig. 1a. b Schematic of the initial transcription experiment (Methods). Above: using TIRFM-based smFRET, we monitored the EFRET variations of the donor–acceptor pair upon NTP addition. The RNAP fluctuates between RPO, ITC6, and ITC > 6, to eventually escape the initiation phase toward the elongation phase, or to release the nascent RNA; below: cartoon that magnifies the interactions between the 5′-RNA end and σ3.2 and the position of the 5′-RNA end. c Fluctuations in the donor (green) and acceptor (red) dyes intensities (above) and the resulting EFRET (below, blue), showing the variation of EFRET from an Unscrunched (US) FRET state, followed by the Partly Scrunched (PS) FRET state upon NTP addition, and ending in the Fully Scrunched (FS) FRET state. Experimental conditions: 200 ms time points (100 ms ALEX, Methods), 500 µM ApA, and 80 µM All NTP. d Similar experiment conducted as described in c, with the RP failing multiple times before reaching the FS FRET state. Experimental conditions: 200 ms time points, 500 µM ApA, and 30 µM of all NTPs and WT promoter. The red solid lines in the lower panels in c and d represent the FRET states extracted from hidden Markov modeling (HMM) (Methods)
