Fig. 7: The formation of G4/R-loop structures decrease the transcription initiation rate in supercoiled DNA by absorbing the negative supercoiling near the promoter.

a Schematic illustration of [FRET2] for the direct observation of the transcription initiation process, featuring Cy3 and Cy5 positioned 4 bp upstream (− 4) and 19 bp downstream (+ 19) from the TSS, respectively. The expected E of DNA is ~ 0.4. Upon transscription initiation, the E is ~ 0.7. When RNAP escapes from the promoter, the expected E is ~ 0.4. b Representative FRET traces of [FRET2] with PQS in non-template. A dashed gray line represents the time of RNAP and NTP addition. c Quantification of FRET peaks observed in 200 s after RNAP injection (0 min), 10 min, and 30 min for NT-PQS and Control (left y-axis with the blue bar and gray bar, respectively). Data points are quantified from 3−4 repetitions with over 200 molecules. Bars represent the average number of events with 95% confidence (black axis). To compare with the fraction of G4/R-loop (or R-loop) from Fig. 6c (right y-axis with a blue circle and gray circle for NT-PQS and Control, respectively), the two graphs are overlaid. The exact data and P-values are provided in the Source data file. ***P  <  0.0005, NS: nonsignificant (two-sided unpaired t test). d Promoter supercoiling density changes (left y-axis with the blue bar and gray bar for PQS and Control, respectively) with the fraction of G4/R-loop (or R-loop) (right y-axis with a blue circle and gray circle for PQS and Control, respectively) over transcription time were calculated by event-driven stochastic simulation. For details on the simulation, refer to Supplementary Fig. 13 and Supplementary document. e Summary schematic of PQS effect on transcription in supercoiled DNA, illustrating the interplay between G4/R-loop formation and transcription initiation events. The supercoiled DNA exhibits a 10-fold enhanced transcription rate due to pre-existing negative supercoiling. As transcription progresses, accumulated negative supercoiling induces transient R-loop formation, which in turn triggers G4 formation, stabilizing the R-loop structure and relaxing the DNA near the promoter. The stable formation of the G4/R-loop hinders RNAP loading due to superhelical relaxation, consequently slowing down the transcription rate.