Figure 4.

Cmr activates transcription of groEL2. (A) Cmr binds at the groEL2 promoter. Radiolabeled groEL2 promoter DNA was incubated with increasing concentrations of Cmr (Rv1675c) before separation of protein-DNA complexes by electrophoresis. Lane 1, no protein; lanes 2–7 contain, 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 μM Cmr, respectively. (B) Cmr activates groEL2 transcription in vitro. A representative autoradiograph is shown. Lane 1, 200 base RNA marker. In vitro transcription reactions contained 0.1 pmole of groEL2 promoter, 1 pmole M. smegmatis RNA polymerase, 40 mM Tris–Cl pH 8.0, 10 mM MgCl₂, 70 mM NaCl, 1 mM EDTA, 1 mM DTT, 250 μg ml⁻¹ bovine serum albumin, 5% glycerol with increasing amounts of Cmr: lanes 2 and 7, 0 μM; lanes 3 and 8, 0.1 μM; lanes 4 and 9, 0.2 μM; lanes 5 and 10, 0.5 μM; and lanes 6 and 11, 1.0 μM. Reactions in lanes 7–11 contained 1 mM cAMP. The groEL2 transcript and the loading control are indicated. (C) The amounts of groEL2 transcript, in the presence (filled bars) or absence (open bars) of 1 mM cAMP, in each of the reactions shown in (B) were quantified using ImageMaster software (GE Healthcare) and plotted as a histogram. Typically 1.0 μM Cmr resulted in ∼3-fold activation of groEL2 transcription over the basal level.