Figure 6.

 Ligand-activated ER promotes transcription reinitiation, whereas p300 enhances transcription initiation. (A) Single and multiple round transcriptional analysis with ER and p300. Chromatin assembly–in vitro transcription reactions were carried out as in Fig. 5B with the indicated components. (Right, single round) Sarkosyl was added 10 sec after initiation of transcription by the addition of rNTPs. Under these conditions, Sarkosyl inhibits transcription reinitiation but not elongation and, therefore, a single round of transcription is obtained. For each reaction condition, the number of rounds of transcription was determined by dividing the amount of transcription in the absence of Sarkosyl by the corresponding amount of transcription in the presence of Sarkosyl. In these experiments, Sarkosyl was used at 0.2% (wt/vol) final concentration, but identical results were observed with 0.05% and 0.4% Sarkosyl (data not shown). The final concentrations of ER, E₂, and p300 in the transcription reactions were 4.5, 30, and 15 nm. (B) p300 enhances the efficiency of transcription but does not promote transcription reinitiation. Reactions were performed with the indicated components, as in A. The final concentrations of ER and E₂ were 4.5 and 30 nm. (C) p300 does not enhance transcriptional elongation. Reactions were performed with the indicated components, as in A. Primer extension analyses were performed with four different primers that yield reverse transcription products of the indicated lengths. The final concentrations of ER, E₂, and p300 in the transcription reactions were 4.5, 30, and 15 nm.