Figure 2.
GTP requirement for 2′,3′-cyclic phosphate ligation. Reaction mixtures containing 50 mM Tris-HCl (pH 8.0), 2 mM MnCl2, 0.1 µM HORNA > p substrate (depicted at bottom), 1 µM wild-type RtcB (top panel) or H337A mutant (bottom panel), and either 0.1 mM GTP (+GTP) or no added nucleotide (−GTP) as specified were incubated at 37°C. The reactions were quenched with EDTA at the times specified. The RNAs were digested with RNase T1 and the products were analyzed by Urea–PAGE and visualized by autoradiography. The 32P-labeled T1 fragments derived from the input 2′,3-cyclic phosphate substrate (HOCUUpC > p), a transient 3′-phosphate derivative (HOCUUpCp) and the ligated product (HOCUUpCpUGp) are indicated on the right.