PMC

(A) Consensus sequence for initial-transcription pausing. Positions are labeled relative to the RNAP-active-center A-site (purple box) and P-site.
(B) Consensus sequence for transcription-elongation pausing. Top, positions numbered relative to RNA 3’ end (pink box). Bottom, positions numbered relative to RNAP-active-center A-site (purple box) and P-site.
(C) Positions of consensus-sequence nucleotides in pre-translocated-state complexes in initial-transcription pausing (ITC-pre) and transcription-elongation pausing (TEC-pre). Grey boxes with white lettering, high-information-content DNA nucleotides of consensus sequence; grey boxes without white lettering; low-information-content nucleotides of consensus-sequence; pink boxes with white lettering, high-information-content RNA nucleotides of consensus sequence. Dotted rectangle, region enlarged in D. Other symbols and colors as in .
(D) Enlarged view, showing unfavorable interaction of RNAP β residue D446 with nontemplate-strand pyrimidine (Y) at A-site (crossed-out bracket labeled βD446).
(E) Positions of consensus-sequence nucleotides in post-translocated-state complexes in initial transcription pausing (ITC-post) and transcription-elongation pausing (TEC-post). Dotted rectangle, region enlarged in F.
(F) Enlarged view showing favorable interaction of RNAP β residue D446 with nontemplate-strand purine (R) at A-site (bracket labeled βD446).

Distribution of pausing sites and DSBs across different cell types. a, pausing intensity in Raji cells relative to pausing sites defined from HeLa cells. Left panel, average mNET-seq coverage measured in Raji cells over pausing sites determined in HeLa cells. Right panel, mNET-seq coverage measured in Raji cells over the pausing sites determined in HeLa cells and divided into quartiles based on pausing site ranks from HeLa cells. Boxes denote 25th and 75th percentiles, center bars show median, and whiskers span from 5% to 95%. The p values were determined by two-sided Mann–Whitney U test. b, DNA breaks mapped in GM13069 at pausing sites established in HeLa cells. Left panel, DSBs measured in untreated GM13069 cells over HeLa-defined pausing sites stratified into four groups based on pausing sites rank from HeLa cells. The p values were calculated using a two-sided Mann–Whitney U test. Right panel, average cumulative profiles of DSBs in GM13069 induced by etoposide treatment at pausing sites established in HeLa cells. p ∼ 0; p values were calculated using a two-sided Wilcoxon signed-rank test for untreated versus each chemical concentration and between concentrations of etoposide.

RNAPII pausing across clusters and cell types. a Distribution of pausing indices across gene promoters in all 31 clusters in our study. Clusters are ordered according to their mean pausing index. The colours in the background denote the level of RNAPII pausing - grey: no pausing, yellow: low pausing, red: mid pausing, dark red: high pausing. b Distribution of expression levels (log2(FPKM+ 1)) of genes in each cluster. The clusters arranged in the same order as in Fig. 5a. The colour background denotes a threshold on expression (log2(FPKM+ 1) =1) as defined in Additional file : Figure S1 – grey: lowly or not expressed, green: expressed. c Four representative clusters displaying four combinations of RNAPII pausing and gene expression. d Distribution of developmental (GO:0045165, GO:0048864, GO:0007498) and cell cycle (GO:0007049) genes across clusters. e RNAPII pausing vs expression for developmental and cell cycle genes (see Methods) across cell types. f Mean pausing indices for diverse gene sets in each cell type. The gene sets were developmental, cell-cycle as well as bivalent, H3K4me3-only and H3K27me3-only genes in ES cells

Overviews of translational pauses found in 1,038 E. coli ORFs by iNP. (A) Venn diagram representation of the occurrence of different pausing categories. Class 1 pausing occurs only in vitro, class 2 pausing occurs only in vivo, and class 3 pausing occurs under both conditions. The numbers indicate the occurrences of each class among the 1,038 genes examined in this study. (B) Classification of the ORFs by the numbers of pausing sites. The number of pausing events in a single gene is shown at the top (0, no pausing; 1, 2, 3, 4, 5 and >5 indicating numbers of occurrence). The numbers below each bar indicate the numbers of genes belonging to the corresponding categories. The aggregate numbers of occurrence in vitro, in vivo, and both in vitro and in vivo are 2,984, 2,541, and 2,015, respectively. (C) Distribution of the estimated pausing strengths in vitro and in vivo. Each pausing event was evaluated for the relative intensity of the polypeptidyl–tRNA signal. The results are shown in the in vitro–in vivo 2D histogram. The position of the major SecM pausing signal is indicated by an arrow. (D) Pausing site placement bias. The relative position of each pausing site within the ORF was scored as a value increasing from the N terminus to the C terminus (). The distribution of the values for different classes of pausing is shown in the histograms.