The presence of a G4 prone sequence upstream of a minimal promoter increases transcriptional activity in the yeast S. cerevisiae

Abstract

Non-canonical secondary structures in DNA are increasingly being revealed as critical players in DNA metabolism, including modulating the accessibility and activity of promoters. These structures comprise the so-called G-quadruplexes (G4s) that are formed from sequences rich in guanine bases. Using a well-defined transcriptional reporter system, we sought to systematically investigate the impact of the presence of G4 structures on transcription in yeast S. cerevisiae. To this aim, different G4 prone sequences were modeled to vary the chance of intramolecular G4 formation, analyzed in vitro by Thioflavin T binding test and circular dichroism and then placed at the yeast ADE2 locus on chromosome XV, downstream and adjacent to a P53 response element (RE) and upstream from a minimal CYC1 promoter and Luciferase 1 (LUC1) reporter gene in isogenic strains. While the minimal CYC1 promoter provides for basal reporter activity, the P53 RE enables LUC1 transactivation under the control of the human P53 family proteins expressed under the inducible GAL1 promoter. Thus, the impact of the different G4 prone sequences on both basal and P53 family proteins dependent expression was measured after shifting the yeast cells onto galactose containing medium. The results showed that the presence of G4 prone sequences upstream of a yeast minimal promoter can increase its basal activity proportionally to their potential to form intramolecular G4 structures; consequently, this improved accessibility, when present near the target binding site of P53 family transcription factors can be exploited in order to regulate the transcriptional activity of P53, P63 and P73 proteins.