Fig. 1: Transcription initiation occurs through dynamic conformational changes.

a Single-molecule measurements of transcription initiation dynamics. The dual-labeled DNA template complexed with Rpo41 and Mtf1 was observed using a total internal reflection fluorescence microscope. b DNA templates used in base-pair-wise measurements of initiation complex dynamics. DNA template I could be stalled at positions +2, +3, +5, and +6, while DNA template II, which differed from DNA template I by four base-pairs (blue), could be stalled at positions +7 and +8. Both templates were labeled with Cy5 at position −16 of the non-template strand (magenta) and Cy3 at position +16 of template strand (green). The transcription promoter (underscored) and start site (arrow) are indicated. c FRET histograms from single-molecule traces with colocalized Cy3 and Cy5 signals at each stalling position. Histograms were fit to single, double, or triple Gaussian peaks (Supplementary Table 1). The brown, green, and magenta curves represent low, mid, and high FRET populations, respectively. Dashed vertical lines mark the major FRET peaks of DNA only, DNA + Rpo41/Mtf1, and the complex at position +7. Dashed lines in magenta mark the major FRET peaks at positions +2, +3, +5, and +6. d Representative smFRET traces at positions 0, +2, and +6 showing the Cy3 (green) and Cy5 (magenta) signals, and the FRET efficiency traces (navy). e The FRET level of the major population in c shown for each stalling position as the center of the major Gaussian peak. The error bars represent the error in finding the peak center position from Gaussian fitting. f The Cy3–Cy5 distance at each stalling position calculated as the average between those obtained from DNA templates I/II (e) and I/II NT (Supplementary Fig. 5). Error bars represent the propagation of the errors in FRET levels.