Interaction Between Transcribing RNA Polymerase and Topoisomerase I Prevents R-loop Formation in E. coli

Abstract

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 co-localized, genome-wide, with RNA polymerase (RNAP) in transcription units. 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, co-localization 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 and in and upstream (within up to 12-15 Kbs) 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 CTD or deletion of EcTopoI domains involved in the interaction is toxic for cells and leads to excessive negative plasmid supercoiling. Moreover, the CTD overexpression leads to R-loops accumulation genome-wide, indicating that the RNAP-EcTopoI interaction is required for prevention of R-loops formation.Article HighlightsTopoI colocalizes genome-wide and interacts with RNAP in E. coliDisruption of the interaction between TopoI and RNAP decreases cells viability, leads to hypernegative DNA supercoiling, and R-loops accumulationTopoI and DNA gyrase are enriched, respectively, upstream and downstream of transcription units in accordance with twin-domain model of Liu and WangTopoI recognizes its cleavage sites through a specific motif and by sensing negative supercoiling

What is a supercoiling-sensitive gene? Insights from topoisomerase I inhibition in the Gram-negative bacterium Dickeya dadantii

“…9A), from 23% (of all differentially expressed genes) in the lowest quartile, up to 65% in the highest quartile. In line with previous results (25), this observation suggests that topoI participates in active transcription, not only at a few highly expressed operons, but as a global mechanism. In contrast, at the transition to stationary phase, the proportion of inhibited vs activated promoters was almost independent of promoter strength (Fig.…”

“…We then investigated a possible relation between neighbouring gene orientations and the response to seconeolitsine. Such a relationship was expected for the same reason as the previous observation, since RNAP-generated supercoils accumulate not only behind actively transcribed genes (25), but more specifically between divergent operons (55), as occurs at the leu promoter of S. enterica where this mechanism was discovered (30,56). The orientation of a gene is here defined by the coding DNA strands of its two neighbours relative to it (in the case of tandem genes, the two neighbours belong to the same strand, which can either be the same as the considered gene or the opposite one).…”

“…Since the leading strand is known to be enriched in highly expressed genes, we looked for a relationship between expression strength and response to seconeolitsine treatment. TopoI is known to colocalise with RNAP and possibly release negative supercoils in its wake at strongly expressed promoters (25), and we therefore expected the latter to be particularly hampered by topoI inhibition. Indeed, we found a very strong and progressive increase in the proportion of inhibited genes depending on their expression level in the exponential phase (Fig.…”

“…However, the steady-state SC level of these strains differs from that of wild-type ones, and the associated transcriptomic response was never monitored. In wild-type cells, topoI seemed a particularly suitable drug target (24), both in clinical research as it is the only enzyme of type IA topoisomerases family in many pathogenic species, but also as a way to study the effect of SC in transcriptional regulation, since this enzyme plays a direct role in the handling of torsional stress associated with transcription (25), while topoIV is predominantly involved in replication (26). Not only was it the first topoisomerase to be discovered (27), but the reduction or loss of its activity due to mutations in the topA gene in E. coli and S. enterica played an important role in the early discovery of the reciprocal interaction between transcription and SC (21, 28–30).…”

Bacterial Argonaute proteins aid cell division in the presence of topoisomerase inhibitors in Escherichia coli

“…We then analyzed smDNA abundance in intergenic DNA regions for divergent, convergent and co-oriented gene pairs. Divergent and convergent gene orientation is associated with negative and positive DNA supercoiling in intergenic regions, respectively, which could affect smDNA production (38). No significant differences in the amounts of smDNAs were detected for the different types of gene pairs for both pAgos (Fig.…”