Radiolabeling and Kinetic Analysis of tRNA Modifications

G₋₁ addition reactions were performed at room temperature by reacting 5′-monophosphorylated [³²P]- tRNA^His (5′-p*tRNA^His) substrate (≤40 nM) with 0.1 mM ATP and 1 mM GTP in the presence of excess enzyme (15 µM). The 5′-p*tRNA^His (with A₇₃ or C₇₃) was generated by in vitro transcription followed by calf intestinal phosphatase (NEB) treatment and labeling with T4 polynucleotide kinase (NEB), as previously described (Jackman and Phizicky 2006a (link)). To measure the k_obs, a saturating amount of enzyme (15 µM SceThg1) was added to a reaction mixture containing p*tRNA^His, 0.1 mM ATP, 1 mM GTP in Thg1 reaction buffer. At specific time points, a 3 µL aliquot was removed from the reaction mixture and quenched by adding 1 mg/mL of RNaseA (Ambion) and 500 mM EDTA. The quenched reaction mixture was incubated at 50°C for 10 min. RNaseA digested samples were treated with 0.5 U calf intestinal phosphatase (CIP) (Invitrogen) and incubated at 37°C for 30 min. The products were resolved and quantified using silica thin-layer chromatography (TLC), as described previously (Jackman and Phizicky 2006a (link); Rao et al. 2011 (link); Smith and Jackman 2012 (link)).