FIG. 6.

Single-round heparin-resistant runoff transcription at the dps promoter carried out with M. smegmatis holo-RNA polymerase. (A) A 10% polyacrylamide gel containing 6 M urea shows an RNA ladder run separately and matched. (B) M. tuberculosis σ^A, σ^F, and σ^H were reconstituted with M. smegmatis core RNA polymerase to obtain the reconstituted holopolymerases. The intensity of each transcript band as obtained from phosphorimager analysis showed the expected transcripts for σ^F (right lane) and σ^H (middle lane). No transcripts were obtained with σ^A-reconstituted RNA polymerase (left lane). (C) In order to test single-round runoff transcription with σ^A-reconstituted RNA polymerase, the rel promoter was used as a positive control. The left lane shows the presence of the transcript, and the right lane shows its absence with rifampin (0.1 μg/ml). (D) Positive controls are shown using the known M. tuberculosis σ^H-dependent sigB promoter in the absence (left lane) and in the presence (right lane) of rifampin, as well as the σ^F-dependent usfXp₁ promoter in the absence (middle lane) and in the presence (right lane) of rifampin. No band was obtained from the M. smegmatis dps promoter in the presence of rifampin with σ^H-reconstituted RNA polymerase (left lane).