Figure 8.

In vitro ribonuclease activity of Mycobacterium tuberculosis VapC26 and comparison of active sites with homologs. When a synthetic RNA containing a fluorophore-quencher pair binds to VapC26, the RNA is digested and the quencher is removed. The released fluorophore generates fluorescence (A and B) Fluorescence measurements, as a function of time, following the addition of Mg²⁺ and Mn²⁺. Based on the results, Mg²⁺ and Mn²⁺ are essential for catalytic activity. VapC26 was treated with EDTA prior to the assay to remove metal ions. Various concentrations of Mg²⁺ and Mn²⁺ were prepared, and 40 units of RiboLock™ (Thermo Scientific) RNase inhibitor was used to prevent contamination. The control contained 50 mM MgCl₂ (or 50 mM MnCl₂), 50 mM Tris-HCl (pH 7.9), 500 mM NaCl, 250 mM Imidazole and 40 units of RiboLock™ (Thermo Scientific) RNase inhibitor. Each experiment was performed in triplicate. (C) Superposition of conserved residues in active sites of VapC26 and its homologs. Residues and metal ions are marked with different colors depending on the family they belonged to.