A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival
Figure 2
MtbGre factor stimulates the cleavage of 20 mer transcript.
(A) Determination of the unit activity of MtbGre. Stalled TEC generated with M. smegmatis RNAP was incubated with different concentrations of MtbGre (10 nM to 5 µM) for 30 mins. Reactions were terminated and resolved on a 20% urea PAGE. (B) Time-course of MtbGre activity. Stalled TECs were incubated at 37°C with 1 µM MtbGre and aliquots were removed at different time points and quenched with urea gel loading dye followed by resolving on a 20% urea PAGE. The time required for 50% cleavage of the TEC was calculated from the plot. (C) The MtbGre homolog-Rv3788 does not induce transcript cleavage. MtbGre could induce the cleavage of +20 nt stalled elongation complex at T7A1 template (lanes 2 and 3). Rv3788 does not have detectable transcript cleavage stimulatory activity (lanes 4 and 5).