FIGURE 3.

RtcB catalyzes intramolecular 3′-phosphate/5′-OH ligation and has a 2′,3′-cyclic phosphodiesterase activity. Reaction mixtures (20 μl) containing 50 mm Tris-HCl (pH 8.0), 0.1 mm GTP, 2 mm MnCl₂, 20 nm ³²P-labeled 20-mer RNA strand with 5′-OH, 5′-phosphate (5′-P), 3′-phosphate (3′-P), or 2′,3′-cyclic phosphate (>P) termini (depicted at the right, with the radiolabeled phosphate denoted by ●), and 200 nm RtcB (where indicated by + above the lanes) were incubated for 30 min at 37 °C. The reactions were quenched by adding EDTA to 4 mm final concentration. Half of the reaction mixture was analyzed directly by denaturing PAGE; the labeled RNAs were visualized by autoradiography (top panel). The positions of the linear substrates, the ligated circles, and multimeric ligation products are indicated on the left. The other half of the reaction mixture was digested with RNase T1. (The relevant sites of RNase T1 incision of the 20-mer substrates and flanking the ligation junction are indicated by arrows above the RNA sequences at the right.) The T1 digests were then resolved by denaturing PAGE and visualized by autoradiography (bottom panel). The positions of the T1 fragments are indicated on the left.