R-loop induced G-quadruplex in non-template promotes transcription by successive R-loop formation

Chun-Ying Lee,Walter Zhao,Christina Mcnerney,Kevin Ma,Ashley Wang,Sua Myong

doi:10.1038/s41467-020-17176-7

Chun-Ying Lee, Walter Zhao + Show 4 more

Open Access

https://doi.org/10.1038/s41467-020-17176-7

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Abstract

G-quadruplex (G4) is a noncanonical secondary structure of DNA or RNA which can enhance or repress gene expression, yet the underlying molecular mechanism remains uncertain. Here we show that when positioned downstream of transcription start site, the orientation of potential G4 forming sequence (PQS), but not the sequence alters transcriptional output. Ensemble in vitro transcription assays indicate that PQS in the non-template increases mRNA production rate and yield. Using sequential single molecule detection stages, we demonstrate that while binding and initiation of T7 RNA polymerase is unchanged, the efficiency of elongation and the final mRNA output is higher when PQS is in the non-template. Strikingly, the enhanced elongation arises from the transcription-induced R-loop formation, which in turn generates G4 structure in the non-template. The G4 stabilized R-loop leads to increased transcription by a mechanism involving successive rounds of R-loop formation.

Highlights

G4-resolving helicases (e.g., BLM, WRN, and XPB/XPD) lead to an altered pattern of G4 formation and transcription factor (TF) binding in the promoter regions[9]
To examine the effect of potential G4-forming sequences (PQS) in transcription, we set up an ensemble in vitro transcription assay in which a reporter probe fluoresces upon binding the transcribed RNA
We have demonstrated that transcription by RNA polymerase (RNAP) on PQS in NT (PQS-NT) induces R-loop formation, which in turn leads to G4 formation in NT strand

Summary

Introduction

G4-resolving helicases (e.g., BLM, WRN, and XPB/XPD) lead to an altered pattern of G4 formation and transcription factor (TF) binding in the promoter regions[9]. PQS is found in the promoter of many potent oncogenes, where it is proposed to regulate the expression of these genes[10]. PQS in the promoter has been shown to regulate gene expression in the presence of G4-binding ligand, which stabilizes G4 structure[12,13,14,15]. Previous studies have shown correlation between PQS at 5′-UTR and transcription level, there is no direct evidence to show whether G4 forms during transcription reaction. Another structure that may arise from transcribing the 5′-UTR.

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