DNA topoisomerase I acts as supercoiling sensor for bacterial transcription elongation

During transcription, RNA polymerase (RNAP) continuously unwinds and rewinds DNA, generating negative and positive supercoils upstream and downstream, respectively. Using single-particle cryo-EM, we elucidated how bacterial RNAP and DNA Topoisomerase I (TopoI), which relaxes negative supercoils, operate in close spatial proximity. TopoI binds to relaxed DNA upstream of RNAP and this involves a conformational switch in the TopoI functional domains. This suggests that TopoI exerts a sensing role prior to the formation of negative supercoils. On DNA substrates mimicking negatively supercoiled DNA, TopoI threads one strand into the active site for cleavage and binds the complementary strand with an auxiliary domain. Transcriptomic and phenotypic analyses suggest that mutations affecting the conformational changes in TopoI impact gene expression and operon polarity in bacteria. In summary, we propose a comprehensive model for DNA relaxation in proximity of active bacterial transcription. In order to study transcriptional changes when TopoI is partially depleted, an E. coli strain encoding a TopoI degradable by a Lon protease system from Mesoplasma florum was engineered and RNA-seq analysis was performed (mf-lon WT, 4h P.I. (AtC) and mf-lon TopA-PDT, 4h P.I. (AtC)). To study transcriptional changes when TopoI variants are expressed, RNA-seq analysis was performed in a strain with TopoI depletion and CRISPR inhibition expressing TopoI variants (mf-lon TopoI-PDT CRISPRi pGB2-TopoI WT or variants (Gly5, G703P and Y704A)).