FIG. 2.

Transcription initiation by recombinant T. aquaticus RNAP. (A) The indicated RNAP core enzymes were combined with recombinant E. coli (Ec) ς⁷⁰ or T. aquaticus (Taq) RpoD in the presence of the T7 A1 promoter-containing DNA fragment, CpA primer, and [α-³²P]UTP substrate. The reaction mixtures were incubated at 45°C for 15 min (lanes 1 to 3 and 4 to 6, respectively), and the products were resolved by denaturing PAGE and revealed by autoradiography. (B) Transcription complexes were formed at the indicated temperatures on the T7 A2 promoter-containing DNA fragment radioactively end labeled on the bottom strand, using recombinant E. coli (Ec) or T. aquaticus (Taq) RNAP holoenzymes, and probed with KMnO₄. Reaction products were resolved on a 6% denaturing polyacrylamide gel and revealed by autoradiography. The permanganate-sensitive bands were assigned using G/A markers (not shown) (14).