FIG. 1.
Rv1222 is a σE anti-sigma factor. (A) Rv1222 inhibits σE-dependent transcription in vitro in a dose-dependent manner. Increasing amounts (1.5, 3, 6, and 12 pmol) of Rv1222 were added to a reaction mixture containing M. smegmatis RNAP and σE. The addition of 3, 6, or 12 pmol of RshA (31) or RslA (5) under conditions where they show activities on their cognate sigma factors had no effect on σE-dependent transcription. DTT, dithiothreitol. (B) Rv1222 interacts with σE. σE was fused to GST, mixed with His-tagged Rv1222, and revealed by immunoblotting with an anti-His antibody. “L” represents a loading control which corresponds to 10% of the reaction mixture prior to any washes, “S” is the supernatant—or unbound Rv1222—recovered upon pelleting the beads immediately before the first wash step, and “P” denotes the bead pellet after all washes. No interaction was detected between GST and Rv1222.