Fig. 6: eRNAs facilitate pause release in a length-dependent manner.

a Schematic of the mammalian pause release assay (top) and the utilized nucleic acid scaffold (bottom). Briefly, a transcription-competent complex that consists of Pol II and a transcription bubble, comprising a template (T-), non-template (NT-) DNA strand a nascent RNA, is assembled (see Fig. 2a). As the DNA contains a 4 nt long G-less cassette succeeded by three Gs (in the non-template strand), pausing can be induced by GTP omission. DSIF and NELF are then added to reconstitute the PEC and to stabilize the pause. The PEC is then isolated by binding the biotinylated DNA non-template strand to magnetic streptavidin-coated beads. Next, eRNAs are added, the supernatant (SN) is removed, and transcription is allowed to resume by the addition of NTPs. b Pause release assay using wild-type NELF and Nr4a1-(a) eRNA fragments (1–50 and 1–200). The left panel shows the urea PAGE analysis for the Nr4a1-(a) 1–50 and 1–200 fragments. Urea PAGE gels for Nr4a1-(a) eRNA (1–100) and two Nr4a1-(a) mutants (102 nt - Δloop 12 mutant; 170 nt - Δloop 1 mutant) are shown in Supplementary Fig. 5b. Samples for all experiments were taken just before NTP addition (0 min) and at different time points after NTP addition (1, 3, 6, 14, and 25 min). The “input” sample contains the PEC sample before its affinity purification using the streptavidin-coated beads. Thus, it allows for the visualization of unbound nascent RNA. For the quantification of pause release efficiency (shown on the right) the intensity of the first transcript elongation band past the pause (boxed in red) was analyzed. c Pause release assay using the NELF patch C mutant and Arc eRNA (1–55, 1–100, and 1–200). The left panel depicts the urea PAGE analysis for the Arc eRNA fragment (1–55), (1–100), and (1–200). Additional data for Nr4a1-(a) and Nr4a1-(b) 1–200 are shown in Supplementary Fig. 5c. The right panel shows the quantification of all experiments, as described for (b). d Pause release assay using the NELF double mutant. Performed under the same conditions as described in (c). Source data for (b–d) are provided in a Source Data file.