Journal: Nucleic Acids Research
Article Title: Oxidative stress rapidly stabilizes promoter-proximal paused Pol II across the human genome
Figure Lengend Snippet: H 2 O 2 inhibits Pol II termination in vitro and increases Pol II engagement in cells. ( A ) Preinitiation complexes were formed on immobilized template DNA in the presence of indicated concentrations of H 2 O 2 . Elongation complexes were pulsed 30 s with limiting α- 32 P-CTP and chased as indicated for 3 min with 500 μM cold ATP, UTP, GTP and CTP. Lengths of limiting-CTP transcripts are indicated; brackets indicate sizes of capped and uncapped transcripts. 6% Urea-PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 100 min with 1 μM flavopiridol, and then 3 min with 0.3 mM H 2 O 2 as indicated. 500 nM triptolide was added either during the last 30 min of flavopiridol treatment, or during all 100 min of flavopiridol treatment. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) 0.1, 1 or 10 μM triptolide was added to preinitiation complexes 30, 10, 3 or 1 min before a 30 s limiting α- 32 P-CTP pulse. Labeled non-tRNA transcripts were quantified, adjusted for loading using cold nuclear RNAs, and normalized to reactions lacking triptolide. Error bars represent S.E.M. from three replicates. ( D ) Preinitiation complexes were formed on immobilized template DNA in the presence of 1 μM flavopiridol alone (C; control) or in combination with 0.3 mM H 2 O 2 (H). Elongation complexes were pulsed 30 s with limiting α- 32 P-CTP and chased 10 min with 500 μM cold ATP, UTP, GTP and CTP. Labeled transcripts from bead-bound (B) and supernatant (S) fractions were quantified to measure termination. Runoff transcripts (508 nt) were considered bound and tRNAs were excluded. Error bars represent S.E.M. and p -values are from 3 replicates (see Gel Appendix). 12% Urea–PAGE. ( E ) Top panel: western blot of RPB1 (sc-55492) and Ponceau S staining. Total (T) or cytosolic (C) and nuclear (N) fractions were obtained from adherent HeLa cells treated 1 h with 0.1% DMSO or 1 μM flavopiridol, and then 10 min with 0.3 mM H 2 O 2 as indicated. Shown are two representative blots for RBP1. The bottom plot pairs with the Ponceau S staining. 4–20% SDS-PAGE. Bottom panel: plot of the percentage of RPB1 in the cytosolic (C/C+N) or nuclear (N/C+N) fractions from five replicates.
Article Snippet: Nuclear walk-on For each reaction, 1–3 × 105 nuclei were diluted to achieve the following reaction conditions ±1.33 μg/ml α-amanitin (Sigma A2263): 20 mM HEPES pH 7.6, 0.5% sarkosyl, 5 mM Mg(C2 H3 O2 )2 , 5 mM DTT, 150 mM KC2 H3 O2 , 0.25 U/μl SUPERase-In and 0.167 μM α-32 P-CTP (PerkinElmer BLU008H001MC).
Techniques: In Vitro, Polyacrylamide Gel Electrophoresis, Labeling, Western Blot, Staining, SDS Page