Fig. 3.

Formation of tripartite supercoiled domains leads to R-loops. (A) Time-trace showing phase (1) negatively supercoiled DNA. In phase (2), as RNAP initiates, it scrunches and unwinds ∼2 turns of DNA, increasing DNA extension by ∼100 nm. After promoter escape, phase (3) elongation proceeds with an unwound bubble of ∼9 bp, and so DNA extension is increased by only ∼50 nm compared with the baseline. Then MfdRA953 binds to RNAP and DNA to form the tripartite supercoiled domain. Ongoing elongation by RNAP causes a gain of positive supercoiling in the external domains, in phase (4), pulling the bead to the surface after passage through the maximal extension state. There follows phase (5), a new state in the DNA extension after the tripartite supercoiled domain event ends. This state, phase (5), is the maximal-extension (i.e., torsionally relaxed) state of the DNA for the extending force used and is consistent with the presence of an R-loop (25). While the DNA is in the maximal-extension state, it is possible for more RNAPs to initiate transcription, and so once the intermediate state ends, the DNA does not always return exactly to a baseline extension but can instead return to a near-baseline extension, phase (1′), indicative of a new ongoing transcription cycle, as we see here. (B) The lifetime of the maximal extension “R-loop” state without RNase HI (n = 29; average = 13,000 ± 3,000 s [SEM]) and with 0.025U/µL RNase HI (n = 17; average = 16 ± 6 s [SEM]); see SI Appendix, Fig. S10 for distributions and fits. (C) Rotation scan of a DNA molecule captured while in the intermediate state, phase (5). A first scan (in red) is taken immediately following formation of the intermediate state [i.e., while the DNA is in the R-loop state, phase (5)], from −42 turns through to +33 turns. Once the DNA becomes positively supercoiled, the R-loop is ejected, and a second scan (blue) is taken from +33 turns to −42 turns. The difference between the two curves in the negative branch of the curve is indicative of an R-loop (25). A rotation scan taken before the experiment began is shown in SI Appendix, Fig. S11. (D) Experimental model. In phase (3), RNAP transcribing on negatively supercoiled DNA is [phase (3′)] bound by Mfd to form the tripartite supercoiled domain. In phase (4), positive supercoiling builds up in the external domain which annihilates the negative supercoiling, increasing end-to-end extension until a maximal extension state is reached. In phase (4′), as net positive supercoils are generated the end-to-end extension again decreases until phase (5) when one of the motors dissociates revealing the R-loop maintained in the DNA.