Fig. 2: EcTopoI is associated with RNAP and with regions of expected transcription-induced negative supercoiling.

a Venn diagram represents an overlap between the EcTopoI and RNAP peaks. b Violin plots of RNAP enrichment in EcTopoI peaks and outer regions (left), and EcTopoI enrichment in RNAP peaks and outer regions (right). The means and medians are indicated by black and blue lines, respectively. Statistically significant differences between means (two-sided t-test, p value = 8e-28 and 9e-123 for RNAP and EcTopoI enrichments, respectively) are indicated by asterisks. c Metagene plot of EcTopoI enrichment within TUs (middle), their upstream (left), and downstream (right) regions. Enrichment is shown for all TUs (black curve), highly-expressed (HETU, orange curve), and least-expressed (LETU, gray curve) sets. The number of TUs in each group is indicated in parentheses. The two insets show zoom-in views of EcTopoI enrichment near transcription start (TU start) and termination (TU end) sites. d Top, metagene plot of RNAP enrichment. Analysis and sets of TUs are the same as in c. Bottom, graphical representation of the Liu & Wang twin-domain model³⁶ showing localization of RNAP and EcTopoI according to the metagene plots in c, d. e Venn diagram represents an overlap between the EcTopoI and RNAP peaks (327 and 2513 peaks, respectively) in cells pretreated with Rif. f Violin plots of RNAP enrichment in EcTopoI peaks and outer regions (left), and of EcTopoI enrichment in RNAP peaks and outer regions (right) for Rif-treated cells (an RpoC ChIP-chip dataset from ref. 44). Means were compared by two-sided t-test. P values 9e-17 and 2e-6 for RNAP and EcTopoI enrichments, respectively, are indicated by asterisks. g Metagene plot of EcTopoI enrichment for cells pretreated with Rif. h Top, metagene plot of RNAP enrichment for cells pretreated with Rif. Bottom, graphical representation showing localization RNAP and EcTopoI according to metagene plots in g, h. i EcTopoI enrichment for HETUs (left) and LETUs (right) in untreated cells (blue bars), cells pretreated with Rif (black bars), and cells overexpressing 14 kDa CTD without Rif (red bars) or followed by Rif treatment (gray bars). Enrichment was quantified for normalized tracks in regions near the transcription start sites (TSS, ± 200 bp from transcription start site), 5 kb upstream regions (US), 5 kb downstream regions (DS), and TU bodies (TU). Enrichments were compared by a two-sided Welch t-test. P values <2e-3 are indicated by asterisks with the number of asterisks indicating the significance level, Bonferroni correction for multiple testing was applied. Bars represent mean values ± SEM, n = 200 TUs for all conditions. j Metagene plot of normalized EcTopoI enrichment for HETUs in cells untreated (blue curve) or pretreated with Rif (red curve). k Metagene plot of normalized RNAP enrichment for HETUs in cells untreated (blue curve) or pretreated with Rif (red curve). l Metagene plot of EcTopoI enrichment for cells overexpressing 14 kDa CTD (top) and a graphical representation of the localization of RNAP and EcTopoI (bottom). m Metagene plot of EcTopoI enrichment for cells overexpressing 14 kDa CTD followed by Rif treatment (top) and a graphical representation of the localization of RNAP and EcTopoI (bottom). n Metagene plot of normalized EcTopoI enrichment for HETUs in cells untreated (blue curve), or treated with Rif (black curve), and in cells overexpressing 14 kDa CTD without Rif (red curve), or followed by Rif treatment (gray curve). Confidence bands (±SEM) in panels j, k, and n are represented by light-colored profiles. Colored areas in panels j and n indicate regions used to quantify enrichment in panel i (US, TSS, TU body, and DS). For ChIP-Seq, fold enrichment is given relative to the input sample.