Opposing roles of two R-loop associated G-quadruplexes in tuning transcription activity

Abstract Guanine (G)-rich sequences in nucleic acids can form non-canonical secondary structures such as R-loops and G-quadruplexes (G4) during transcription. The R-loop formed on the template strand promotes and stabilizes G4 in the non-template strand. However, the precise role of G4/R-loop-forming sequences on transcription remains poorly understood. In this study, we investigated the effect of different potential G4-forming sequences (PQSs) on G4/R-loop formation and transcription dynamics. We employed gel-based assays and single-molecule fluorescence resonance energy transfer (smFRET) to measure RNA synthesis and concomitant formation of G4 and R-loop during transcription by T7 RNA polymerase. We reveal two types of R-loop that form successively; an R-loop with an intramolecular DNA G4 (IG4) initially forms during transcription, followed by an R-loop with an intermolecular DNA:RNA hybrid G4 (HG4). We found that IG4 R-loops inhibit, whereas HG4 R-loops enhance transcription. We identified that an HG4/IG4 ratio highly correlates with transcriptional activity. PQS with short linkers favors IG4, reducing transcription, while PQS with long linkers that induce loosely folded PQS favor HG4, increasing transcription. Since IG4 formation precedes HG4, tightly folded PQS forms IG4 quickly and stably, slowing its conversion to HG4 and reducing transcriptional enhancement.