Bacterial topoisomerase I (TopoI) removes excessive negative supercoiling and is thought to relax DNA molecules during transcription, replication and other processes. Using ChIP-Seq, we show that TopoI of Escherichia coli (EcTopoI) is colocalized, genome-wide, with transcribing RNA polymerase (RNAP). Treatment with transcription elongation inhibitor rifampicin leads to EcTopoI relocation to promoter regions, where RNAP also accumulates. When a 14 kDa RNAP-binding EcTopoI C-terminal domain (CTD) is overexpressed, colocalization of EcTopoI and RNAP along the transcription units is reduced. Pull-down experiments directly show that the two enzymes interact in vivo. Using ChIP-Seq and Topo-Seq, we demonstrate that EcTopoI is enriched upstream (within up to 12-15 kb) of highly-active transcription units, indicating that EcTopoI relaxes negative supercoiling generated by transcription. Uncoupling of the RNAP:EcTopoI interaction by either overexpression of EcTopoI competitor (CTD or inactive EcTopoI Y319F mutant) or deletion of EcTopoI domains involved in the interaction is toxic for cells and leads to excessive negative plasmid supercoiling. Moreover, uncoupling of the RNAP:EcTopoI interaction leads to R-loops accumulation genome-wide, indicating that this interaction is required for prevention of R-loops formation.