FIG. 2.
The Pminor transcriptional start is identified. In vitro transcription reactions were assembled by adding 1.95 μl of a solution containing reconstituted polymerase (0.2 pmol core plus 0.5 pmol of σ70) in protein buffer I to 0.02 pmol of linear DNA in 2.05 μl of DNA buffer I. (A) Transcription was initiated by adding 1 μl of NTP mix to the protein-DNA mix containing Pminor DNA. As indicated, each NTP was added at the following concentrations: 1 mM UTP and 0.25 mM each ATP and GTP. The specific activity of [γ-32P]GTP or [γ-32P]ATP (where indicated by the asterisk) was 7 × 105 dpm/pmol. Assignments of labeled RNA products are shown. The black arrow signifies 5-nt products, consistent with the migration of the pppACN3 marker (not shown) kindly provided by N. Nossal. (B) Denaturing acrylamide gel showing the products of primer extension assays next to a Pminor DNA sequencing ladder. The unlabeled Pminor, P+1C, and P2 transcripts were generated by multiple-round transcription assays, which were initiated by the addition of 1 μl of NTP mix II. (C) Single-round transcription was initiated by adding 1 μl of NTP mix I with heparin. Transcripts arising from the Pminor, P+1C, and P2 promoters are indicated.