FIGURE 2.

RtcB seals 3′-monophosphate/5-OH RNA breaks. Reaction mixtures (10 μl) containing 50 mm Tris-HCl (pH 8.0), 0.1 mm ATP, 0.1 mm GTP, 2 mm DTT, 0.1 mm spermidine, 2 mm MnCl₂ (for E. coli RtcB; lanes 2, 4, 6, and 8) or 10 mm MgCl₂ (for plant tRNA ligase, AtRNL; lanes 3, 6, 9, and 12), 20 nm ³²P-labeled broken RNA stem-loop substrates as specified (formed by mixing 20 nm labeled ³²P-20-mer with 200 nm unlabeled complementary 20-mer to yield the substrates illustrated at the bottom), and 200 nm RtcB or AtRNL (where indicated by + above the lanes) were incubated for 30 min at 37 °C. The products were resolved by denaturing PAGE and visualized by autoradiography. The radiolabeled RNAs corresponding to the 20-mer substrate strand and the ligated hairpin stem-loop are indicated on the left.