Fig. 2: Classification of σ70-dependent transcription pauses.
a Example of σ70-dependent pause upstream of the yjcE coding sequence (CDS) identified by RNET-seq in the σ70-ΔgreAB strain. The genomic coordinates for 3’ ends of all uniquely mapped RNA reads (bottom lane) were determined and the read count for each 3′ end position was calculated and plotted (top lane). The genomic positions where 3’ end/3’ end median (51-bp window) read counts ratio (pause score) was ≥ 20 and read counts/106 reads was ≥ 10 satisfied our stringent definition for a pause site. b Venn diagrams show the total and shared numbers of pauses identified in σ70-WT (n = 7412), β’-WT (n = 3543), σ70-ΔgreAB (n = 12211) and β’-ΔgreAB (n = 6498) strains. c Distribution of σ70-dependent pauses among CDS, UTR, Antisense, tRNA, rRNA and ncRNA regions in σ70-WT and σ70-ΔgreAB strains. The “Antisense” pauses included those in CDS, tRNA, rRNA and ncRNA genes. d Distribution of pause sites in promoter-proximal regions. The TSS coordinates identified by dRNA-seq64 were used to plot pause counts against the pause distance from the nearest TSS on the same DNA strand. The zero and positive coordinates correspond to the pauses overlapping the TSS or located downstream of the TSS, respectively. The upper panel shows the counts of pauses in 50-nt bins within −2000/+2000-bp window centered at the TSS. The bottom panel shows the ratio obtained by dividing the count of pause sites in a 5-bp sliding window to the total count of pause sites in the −50/+200-bp register surrounding the TSS. Heatmap (e) and mean (f) of the read counts for σ70-ΔgreAB G1 pause sites (n = 3099) in σ70-ΔgreAB (left) and σ70-WT (right) strains. The pause sites were ranked based on the pause score (described in a). The counts of reads aligned to the sense and antisense strands in each coordinate were normalized to 0 to 1 and 0 to −1 by dividing the maximum read count in each −50/+200-bp region. The regions with multiple pause sites were counted only once (e). The dashed line and number on the top indicate the distance of the peak from the TSS. The line and the shadowed region represent the mean and 95% confidence interval for the read counts ratio (f).
