Figure 2.

Downstream DNA is dispensable for transcription initiation by RNA polymerase II under conditions that bypass the requirement for TFIIH in initiation. (A) Schematic diagram of reaction protocol. BF's, basal initiation factors; *CTP, [α-³²P]CTP; NTP's, ribonucleoside triphosphates; RNAPII, RNA polymerase II. (B) AdML (lanes 1–4) or Ad(−9/−1) (lanes 5–12) DNA templates were incubated for 20 min at 30°C with or without 15 units of PstI. Preinitiation complexes were assembled, with or without TFIIH, on templates as described in Materials and Methods. Abortive initiation assays (A.I.) were performed with 5 μM dATP, 200 μM CpU, and 0.5 μM [α-³²P]CTP (3000 Ci/mmol). Promoter escape assays (P.E.) were performed with 5 μM ATP, 200 μM CpU, 0.5 μM [α-³²P]CTP, 100 μM UTP, and 150 μM 3′-O-MeGTP. (C) Shorter exposure of the lower portion of the gel shown in B to allow visualization of abortively initiated trinucleotide transcripts in lanes 5–12.