Figure 3.

PTRF stimulates transcription on tailed templates. (A) PTRF-mediated transcript release and transcriptional stimulation. Reactions contained 0.5 U Pol I, immobilized tailed template (pCAT-T6-T₁), 30 ng TTFΔN185 and 1, 3 or 10 ng histidine-tagged PTRF as indicated. Transcripts were separated into template-bound (b) and released (r) fractions by magnetic attraction. (B) Time-dependent transcript release and transcriptional stimulation. Transcription assays contained 100 ng of immobilized tailed template pCAT-T6-T₁, 0.5 U mouse Pol I and 30 ng TTFΔN185. After incubation for 10 min at 30°C, 3 ng PTRF was added and incubation was continued for another 1 or 9 min. Transcripts were fractionated into template-bound (b) and released (r) molecules. (C) Transcript release on ternary transcription complexes. Transcription reactions contained bead-bound tailed template (pCAT-T6-T₁), NTPs (including [α-³²P]GTP), 0.5 U Pol I and 30 ng recombinant TTF-I. After incubation for 10 min, complexes paused at the terminator were isolated by magnetic attraction, washed with buffer AM-200 and incubated for another 10 min with 10 ng PTRF in the presence of cold NTPs.