Fig. 2. In vitro transcription assays of defined mononucleosomal templates.

A. Expected products for the d19B, d39B, and d134B templates in which the mononucleosome has been placed at defined positions (d:19 bp, 39 bp, 134 bp) relative to the TSS. Two products are shown for each: one resulting from Pol II pausing at a previously mapped site (P) within the nucleosome and the other from transcription through the entire nucleosome (FL, full-length).

B. Indicated templates (chromatinized or in naked DNA form) were transcribed in vitro in the presence of purified Pol II, the GTFs (TFIIA, B, D, E, F, H), and the cofactor PC4. All reactions contained HNF4α and Mediator. Complete complement (PBAF, p300, FACT, SII) of chromatin cofactors (chrom cofs), as well as acetyl CoA (10 μM), was added to reactions containing chromatin templates (lanes 3, 6, 9). Product sizes for the d19B and d39B templates are indicated to the left; product sizes for the d134B template are indicated to the right. Following PIC assembly in the presence of the factors, transcription was allowed to proceed by adding labeled NTPs for 60 min. Uniformly radiolabeled RNA products were analyzed by electrophoresis. Numbers underneath show relative transcription levels (rel txn) of the full-length (FL) RNA. Transcriptional levels were separately normalized for each template. See also Fig. S2 and S3.

C. In vitro transcription analysis of the chromatinized d39B template. Reactions containing the template were reconstituted and analyzed as in (B) except that all reactions contained acetyl CoA and Mediator and chromatin cofactors (PBAF, p300, FACT, SII) were added as indicated. The full-length (FL, 219 ntd) and paused (P, ~85 ntd) transcripts generated by this template are identified. Percent read-through (RT) is also shown. Asterisks in lanes 3 and 4 signify that the 85-mer could not be accurately quantified (“n.d.”) and that RT levels are visual estimates.