Fig. 5. Uncoupling of RNAP:EcTopoI complex is toxic for cells, leads to hypernegative supercoiling of plasmids, and accumulation of R-loops.

a Growth curves for E. coli DY330 topA-SPA harboring pCA24 GFP (i), pCA24 14 kDa CTD (ii), or pCA24 topA (iii) plasmids. Data for induced (+IPTG 1 mM) and non-induced (−IPTG) cultures are shown. Shade represents a 0.95 confidential interval of the mean of three biological replicates. Gray lines mark aliquots collection for plasmid extraction. b Quantification of cell length in CTD or GFP producing cultures (left). Cells >10 µm are collected into an overflowing bin. The number of cells is indicated in parentheses. Representative fields are shown on the right. c Graphical representation of truncated versions of a topA gene constructed by recombineering in E. coli BW25113. d Growth curves of E. coli BW25113 strains with truncated versions of topA and the wild-type control (left). Shade represents 0.95 confidential intervals of the mean. Quantification of doubling time for exponential regions of growth curves (right). e Quantification of cell length for E. coli BW25113 strains with truncated versions of topA and the wild-type (left). Vertical dashed line marks 2*mean cell length for wild-type. Representative fields are shown on the right. f Mutations in gyrase genes (gyrA, gyrB) found in E. coli BW25113 topAΔ30 clones. An asterisk indicates amplification of a chromosomal region containing TopoIV genes; clones lacking compensatory mutations are highlighted in green. g Supercoiling of pCA24 GFP (i), pCA24 topA (ii), and pCA24 14 kDa CTD (iii) plasmids extracted from exponentially growing E. coli DY330 topA-SPA. Time-points correspond to panel a. Supercoiling of pCA24 GFP (iv) plasmid extracted from exponentially growing E. coli BW25113 topAΔ30 (time-course, on the left) or E. coli BW25113 wt (two rightmost lanes). (v) Supercoiling level of the pCA24 GFP plasmid extracted from overnight cultures of different clones of E. coli BW25113 topA mutants and from the wild-type control. Clone numbers correspond to panel f. Nic - nicked plasmid, L - linear plasmid, −sc - negatively supercoiled plasmid, HCF - hypercompacted plasmid. h Metagene plots of normalized strand-specific read coverage depth obtained in DRIP-Seq experiments for E. coli DY330 topA-SPA for HETU (upper panel, rRNA operons were excluded) and LETU (lower panel) sets. Schematic TUs are shown below. Data for CTD-/Rif- condition are shown with a dashed line, coverage depths for the coding and template strands are indicated by dark-red and dark-blue fillings, respectively. Data for CTD+/Rif− condition are shown with a solid line, coverage depths for the coding, and template strands are indicated by light-red and light-blue fillings, respectively. i DRIP-Seq data for pCA24 14 kDa CTD for CTD+/Rif− and CTD+/Rif+ conditions and corresponding RNase HI-treated controls. Coverage depths for “−” and “+” strands are shown in light-blue and light-red, respectively. A linearized map of the plasmid is shown below. Source data are provided as a Source Data file.