Fig. 2.

Human CE promotes cotranscriptional RLF. (A) Template DNA for in vitro transcription driven by T7 promoter. Plasmid DNA was linearized by restriction enzyme digestion. The length of runoff transcripts is 295 nt. (B) The 1.2 μg of purified human CE was resolved by SDS/PAGE and visualized by Coomassie staining. (C) CE promotes the formation of HMW transcripts. In vitro transcription with CTDp-T7 RNAP was performed with increasing amounts (0, 60, 150, and 300 ng) of CE (lanes 1–4). Transcription by CTDun-T7 RNAP with increasing amounts (0, 60, 150, and 300 ng) of CE is shown in lanes 5–8. Transcription by T7 RNAP with increasing amounts (0, 60, and 300 ng) of CE is shown in lanes 9–11. CE-independent and CE-dependent HMW are indicated. The arrow indicates runoff transcripts. (D) HMW transcripts are DNA·RNA hybrids. Transcription of CTDp-T7 RNAP was performed in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of 300 ng of CE. Transcription was terminated by heat (70°C, 15 min). Subsequently reaction mixtures were treated with (lanes 2 and 4) or without (lanes 1 and 3) RNase H, and RNAs were resolved by denaturing PAGE.