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. Author manuscript; available in PMC: 2014 Oct 30.
Published in final edited form as: Trends Cell Mol Biol. 2013;8:13–23.

Figure 1. 603 nucleosome forms a barrier to transcribing T7 RNAP.

Figure 1

A. The schematic diagram of the template and the experimental approach for analysis of transcription of nucleosomal templates by T7 RNAP. The locations of the 603 nucleosome-positioning sequence (NPS, dotted oval) and the T7 promoter are shown. After transcription initiation, T7 RNAP was stalled at the position -5 relative to the boundary of 603 nucleosome forming EC-5. The transcripts were pulse-labeled. Then transcription was continued using unlabeled NTPs at 40 mM KCl and terminated after different time periods (from 10 to 180 seconds). Labeled RNA was analyzed by denaturing PAGE. B. Analysis of labeled transcripts by denaturing PAGE. The locations of the nucleosome (oval), the nucleosome dyad (square), the EC-5, position +49 and the run-off transcripts are shown. Dashed line: nucleosome-specific pausing. M: MspI digest of pBR322 plasmid. Note that a considerable fraction of elongation complexes completes transcription after 180 seconds, suggesting that the nucleosomal barrier is relatively low.