Assessingin vivothe impact of gene context on transcription through DNA supercoiling

Gene context can have significant impact on gene expression but is currently not integrated in quantitative models of gene regulation despite known biophysical principles and quantitativein vitromeasurements. Conceptually, the simplest gene context consists of a single gene framed by two topological barriers, known as the twin transcriptional-loop model, which illustrates the interplay between transcription and DNA supercoiling.In vivo, DNA supercoiling is additionally modulated by topoisomerases, whose modus operandi remains to be quantified. Here, we bridge the gap between theory andin vivoproperties by realizing inEscherichia colithe twin transcriptional-loop model and by measuring how gene expression varies with promoters and distances to the topological barriers. We find that gene expression depends on the distance to the upstream barrier but not to the downstream barrier, with a promoter-dependent intensity. We rationalize these findings with a first-principle biophysical model of DNA transcription. Our results are explained if TopoI and gyrase both act specifically, respectively upstream and downstream of the gene, with antagonistic effects of TopoI, which can repress initiation while facilitating elongation. Altogether, our work sets the foundations for a systematic and quantitative description of the impact of gene context on gene regulation.