Fig. 1.

Cotranscriptional capping in stalled transcription complexes. (A) Ternary complexes were incubated with 12 ng of CE and 50 μM GTP at 30°C. Capping reactions (lanes 1-5 for +16, lanes 6-10 for +24, and lanes 11-15 for +42) were stopped at the times shown. Products were subjected to phenol/chloroform extraction, ethanol precipitation, and PAGE/8 M urea, followed by autoradiography. The two small spots in lane 6 (above +24) are RNAs produced by slippage of RNAPII (30). (B) To confirm cap formation on RNA, +24 stalled complexes were incubated with [α-³²P]GTP with and without CE (120 ng). RNAs (24 nt) were isolated, treated with P1 nuclease and AP, and analyzed by TLC. Lane 1, untreated RNA; lanes 2 and 3, capped and uncapped RNAs digested with P1 nuclease and AP. The position of authentic GpppA run in parallel is indicated.