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. 2004 May 10;101(20):7572–7577. doi: 10.1073/pnas.0401493101

Fig. 5.

Fig. 5.

CE can overcome NELF-mediated transcription repression. (A) PICs were assembled with purified GTFs on a linear DNA by incubation in TB60 for 20 min at 30°C. DSIF (30 ng), NELF, and CE were then added as shown, and incubations continued for 10 min. Transcription reactions were chased with a mixture of 600 μM ATP, CTP, and GTP; 0.3 μM [α-32P]UTP; 2 μM UTP and 16 units of RNasin for 6 min at 30°C. Reactions were stopped with stop buffer, extracted with phenol-chloroform, ethanol precipitated, and analyzed by 7% PAGE/8 M urea and autoradiography. PICs were chased with either NTPs alone (lane 1) or first incubated with DSIF (lane 2) or NELF (lane 3) or both (lane 4) and chased with NTPs. PICs incubated with both DSIF and NELF were further incubated with 0.12, 12, or 120 ng of CE (lanes 5-7) and chased with NTPs. For lanes 8 and 9, PICs were chased with NTPs in the presence of CE alone (12 and 120 ng). The dots indicate the approximate positions of transcripts of 42 and ≈200 nt. (B)Asin A, except that transcription reactions were chased with NTPs (50 μM ATP/5 μM CTP/0.3 μM [α-32P]UTP/50 μM 3′-OmeGTP/1 μM UTP/16 units of RNasin) for 5 min to produce +24 stalled transcripts, which were analyzed by 18% PAGE in 8M urea.