FIG. 2.

Δpgd1 and Δsin4 mutants have a general defect in plasmid multiround transcription. PICs were formed on plasmid template for 40 min, NTPs were added to initiate transcription, and the reactions were stopped after 2.5 (A) or 40 (B) min. Transcripts were assayed by primer extension (gels). Results of typical transcription reactions with wild-type, Δpgd1, and Δsin4 extracts and with no activator (−), Gal4-AH (A), Gal4-VP16 (V), or Gal4-Gcn4 (G) are shown. Levels of activation (n-fold) are indicated below the gels. Data in the graphs are averages from at least four experiments, with the level of mutant transcription calculated as a percentage of wild-type transcription. (C) Rounds of transcription were calculated by dividing multiround transcription by single-round transcription. The results shown are an average of three experiments. (D) VP16-activated multiround transcription was assayed as in panel B with 0, 1, or 2 μl of purified Pol II-Med complex (∼0.04 pmol/μl) added to the mutant nuclear extracts (NEs).