Figure 4.

The requirements for TFIIH, an ATP(dATP) cofactor, and downstream DNA in promoter escape by RNA polymerase II are bypassed after treatment of very early elongation intermediates with Sarkosyl. (A) Transcription reactions were performed with the premelted Ad(−9/−1) promoter, with a pulse–chase protocol (diagrammed at the top of the figure) to test the requirement for TFIIH specifically during promoter escape. Preinitiation complexes were assembled on the Ad(−9/−1) promoter, with or without TFIIH, as described in Materials and Methods. Short RNA transcripts were synthesized during a 20-min incubation at 30°C with 5 μM ATP, 200 μM CpU, 0.5 μM [α-³²P]CTP, and 0.5 nM UTP. Where indicated, Sarksoyl was added to a final concentration of 0.1% (wt/vol). One minute later, short transcripts were chased into longer transcripts in the presence of 100 μM ATP or 100 μM ATPγS and 200 μM CTP, 100 μM UTP, and 150 μM 3′-O-MeGTP. (B) Preinitiation complexes were assembled on the duplex AdML DNA template as described in Materials and Methods. After synthesis of short radioactively labeled transcripts according to the procedure described for A, reaction mixtures were incubated for 20 min with or without 5 units of HindIII. Sarkosyl was added to reaction mixtures at the concentrations indicated in the figure, and short transcripts were chased into longer transcripts as described in the experiment in A.