FIG. 5.
Primer extension analysis of the ctaA promoter determines two transcriptional start sites. The end-labeled primer (FMH255) was annealed to RNA and then extended with reverse transcriptase. In lane 1, mRNA was synthesized in an in vitro transcription reaction mixture containing ResD∼P, the DNA template (EcoRI fragment of pXH43) (Fig. 3B, section 1), and RNAP isolated from cells at stage T4 in SSG medium. In lane 2, mRNA was synthesized in an in vitro transcription reaction mixture containing ResD∼P, the DNA template (EcoRI fragment of pXH43) (Fig. 3B), and RNAP isolated from cells at stage T0 in SSG medium. Lanes A, T, C, and G contain sequencing ladders generated by annealing the same end-labeled primer to a plasmid (pXH43) containing the 5′ end of ctaA and extending it with Sequenase (United States Biochemical Corp.). The sequence of the region is indicated at the right. The asterisks indicate the base to which the primer extension products map.