Gene Expression Modulated by G4, R-loop and DNA Supercoiling

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PROJECT SUMMARY G-qudruplex (G4) is a noncanonical secondary structure that can form in both DNA and RNA. Human genome contains over 400,000 potential G4 forming sequences (PQS) and they are highly enriched in upstream of oncogene promoters and regulatory genes, strongly suggesting a switch-like function with programed positioning. PQS is also prevalent in e. coli genome, located in important regulatory regions. Indeed, many studies have demonstrated the role of G4 in up or downregulating genomic processes including replication, transcription and translation. Our recent study demonstrated that in transcription, G4 forming sequence located in the non- template strand leads to a robust formation of R-loop (mRNA annealed to template strand), which in turn, induces G4 structure in the non-template strand. Remarkably, such R-loop/G4 structure drives enhanced transcription by a mechanism that involves successive formation and release of R-loop. We show that when positioned in a plasmid i.e under torsional constraint, such G4/R-loop structure can tune the transcription activity up or down depending on the distance from the transcription start site. Furthermore, 5’UTR-G4 bearing mRNA (RG4) leads to over 10-fold enhanced translation in a cell-free system and in e.coli cells. Upon testing several plausible hypotheses, we propose that the RG4 structure promotes translation by blocking ribosomes from sliding off the mRNA. Building on these exciting new findings, we propose to investigate the impact of G4, R-loop and supercoiling in transcription and translation primarily in T7 RNAP system and in e. coli cells. By combining quantitative biochemical tools and newly developed single molecule platforms suited to measure stepwise progression of transcription in a linear or plasmid DNA, we will examine how different sequence, length and position of G4 forming sequence leads to R-loop formation, mRNA output and protein production. Accomplishing the proposed goals will reveal the structure-function relationship of how G4, R-loop and supercoiling regulates transcription and translation activity.