Figure 5.

TFIIE and TFIIH enhance the extent of promoter escape. (A) TFIIE and TFIIH have a minimal effect on the rate of RNA synthesis. Transcription reactions were assembled and initiated in a large volume as indicated in the schematic. (B) TFIIE and TFIIH greatly enhance the extent of RNA synthesis. Transcription reactions were performed as in A, both in the presence and absence of TFIIE and TFIIH. The RNA products in lanes 1 and 3 result from 20 min of transcription, whereas those in lanes 2 and 4 result from 30 min of transcription. (C) TFIIE, TFIIH, and dATP enhance the extent of transcription by affecting promoter escape. Reactions were performed according to the procedure diagrammed beside the figure. Note that dATP was added with the nucleotides rather than with the proteins. AMP (1 mM) was added as the initiating nucleotide along with UTP (625 μM), CTP (25 μM [α-³²P]CTP, 5 μCi per reaction), and, where indicated, dATP (5 μM). The 15-nt RNA product was resolved by 14% denaturing PAGE.