FIGURE 5.

In vitro reconstitution and transcription activity of the ω₆-bearing RNAP holoenzyme. Shown are the subunit compositions of reconstituted core RNAP with ω₆ on 8–20% gradient SDS-PAGE (A) and reconstituted holo-RNAP with ω₆ on10% SDS-PAGE (B). C, promoter DNA-polymerase complex formation. Lanes 1–4 show the indicated concentration of reconstituted ω₆RNAP forming a heparin-resistant DNA-protein complex; lane 5 shows the complex formed by reconstituted wild-type polymerase. The free DNA and DNA-protein complex are marked with arrows. D, T7A1 promoter-specific in vitro single round transcription assay generating transcript with purified RNAP holoenzyme isolated from cells (WT) and holoenzyme versions of reconstituted ωRNAP and ω₆RNAP. Enzyme and promoter concentrations used are 2 and 0.2 pmol, respectively. E, abortive transcription assay detecting first phosphodiester bond formation by 20% urea-PAGE. F, transcription activity of ωRNAP/ω₆RNAP on preformed elongation complex with synthetic coding strand and 9-mer RNA primer along with bound enzyme. Lanes 1 and 3, with additional nucleotide added forming 11-mer transcript; lanes 2 and 4, no addition of nucleotide showing 9-mer RNA primer in elongation complex with template DNA strand and bound enzyme.