Abstract
Movement of the transcription machinery along a template alters DNA topology resulting in the accumulation of supercoils in DNA. The positive supercoils generated ahead of transcribing RNA polymerase (RNAP) and the negative supercoils accumulating behind impose severe topological constraints impeding transcription process. Previous studies have implied the role of topoisomerases in the removal of torsional stress and the maintenance of template topology but the in vivo interaction of functionally distinct topoisomerases with heterogeneous chromosomal territories is not deciphered. Moreover, how the transcription-induced supercoils influence the genome-wide recruitment of DNA topoisomerases remains to be explored in bacteria. Using ChIP-Seq, we show the genome-wide occupancy profile of both topoisomerase I and DNA gyrase in conjunction with RNAP in Mycobacterium tuberculosis taking advantage of minimal topoisomerase representation in the organism. The study unveils the first in vivo genome-wide interaction of both the topoisomerases with the genomic regions and establishes that transcription-induced supercoils govern their recruitment at genomic sites. Distribution profiles revealed co-localization of RNAP and the two topoisomerases on the active transcriptional units (TUs). At a given locus, topoisomerase I and DNA gyrase were localized behind and ahead of RNAP, respectively, correlating with the twin-supercoiled domains generated. The recruitment of topoisomerases was higher at the genomic loci with higher transcriptional activity and/or at regions under high torsional stress compared to silent genomic loci. Importantly, the occupancy of DNA gyrase, sole type II topoisomerase in Mtb, near the Ter domain of the Mtb chromosome validates its function as a decatenase.
Highlights
Translocation of the transcription machinery along the duplex DNA molecule causes axial rotation of the DNA duplex relative to the RNA polymerase (RNAP) complex
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
Genetic and in vitro studies in Escherichia coli have implicated that negative supercoils are acted upon by relaxases i.e. Topo I (Type I) and Topo IV (Type II) while the positive supercoils are removed by the activities of DNA gyrase and Topo IV [2,3,4]
Summary
Translocation of the transcription machinery along the duplex DNA molecule causes axial rotation of the DNA duplex relative to the RNAP complex. The genome-wide gene expression studies have linked the respective relaxation and supercoiling activities of Topo I and DNA gyrase in the modulation of gene expression profile, highlighting the global regulatory roles of topoisomerases [14,15,16]. The mechanism of their in vivo influence on gene expression is not understood due to inadequate information about their interaction with the genome. Direct demonstration of their occupancy on different genomic territories is not yet carried out which is a pre-requisite in determining their impact on supercoiling gradients generated in transcriptionally active regions spanning whole genome. In order to achieve such comprehensive view of topoisomerase dynamics, function and factors affecting their distribution, we have deciphered in vivo genome-wide binding profile of both classes of topoisomerases together taking the advantage of non-redundancy of topoisomerases in Mycobacterium tuberculosis (Mtb) genome
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