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Fig. 4 | Genome Biology

Fig. 4

From: Alternative DNA secondary structure formation affects RNA polymerase II promoter-proximal pausing in human

Fig. 4

Single-nucleotide resolution signals of mNET-seq demonstrate highly stable secondary structures located upstream of the paused sites. a Heat map representations of mNET-seq coverage (black), secondary structure free energy (green-red), and GRO-seq coverage (blue) for Pol II bound genes in HeLa-S3 cells are shown. Pol II-bound genes were ordered by the distance of the strongest mNET-seq read spike to each gene’s TSS. A high magnification of a section of the heat maps displays a thin, bright line of relatively low secondary structure free energies (marked by arrows). b An average free energy profile (purple) and average Pol II coverage (red) centered at the strongest mNET-seq read spike (black) are shown (n = 10,428). At the average free energy of − 3.25 kcal/mol, DNA secondary structures are, on average, about 10 to 40 nt upstream of the peak of the highest mNET-seq read spikes. c Schematic representation of genic and intergenic pausing sites relative to annotated gene transcription start and termination sites. Cumulative distribution of distances between pausing sites to the TSS or TTS: pausing sites within human RefSeq annotated genes (to TSS, green), and pausing sites located outside of human annotated genes (to TSS (red) and to TTS (blue)). d Average profile plot of mNET-seq coverage (black) and secondary structure free energy (purple) at Pol II pausing sites that are located within annotated human genes and centered on the highest mNET-seq read spike are shown (top panel). At the average free energy of − 3.2 kcal/mol, DNA secondary structures are, on average, about 24 to 48 nt upstream of the peak of the highest mNET-seq read spikes. Average profile plot of mNET-seq coverage (black) and secondary structure free energy (purple) at Pol II pausing sites that are not located within annotated human genes and centered on the highest mNET-seq read spike are shown. At the average free energy of − 2.8 kcal/mol, DNA secondary structures are, on average, about 24 to 52 nt upstream of the peak of the highest mNET-seq read spikes (bottom panel). The Mfold analysis was used here, and the ViennaRNA anlysis is shown in Additional file 1: Figure S5

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