High frequency transcription leads to rapid R-loop formation.

R-loops are transcriptionally generated three-stranded nucleic acid structures where the mRNA hybridizes with template DNA, leaving a displaced single-stranded non-template DNA loop. Previously, we demonstrated that R-loop and subsequent G-quadruplex formation upregulate transcription. However, the mechanistic basis of how transcription activity generates R-loop formation is unknown. Here, we investigate the kinetics of transcription and its impact on R-loop formation using single-molecule fluorescence resonance energy transfer (smFRET) and electrophoretic mobility shift assay (EMSA). We show that R-loop formation is tuned by the frequency and the rate of transcription, controlled by the RNAP and NTP concentrations, respectively. We provide a plausible mechanism in which gradually increasing the duration of the promoter opening leads to the R-loop formation. Through stochastic simulation, we demonstrate that the frequency of transcription primarily governs R-loop formation. This work highlights the intricate balance between transcription dynamics and R-loop formation, providing new insights into the structure-function relationship.