Molecular mechanism of substrate recognition and specificity of tRNA^His guanylyltransferase during nucleotide addition in the 3′–5′ direction

Construction of two-piece tRNAs. (A) Two-piece tRNA variants are drawn as cloverleaf structures. S. cerevisiae tRNA^Phe with GAA anticodon altered to GUG (SctRNA^Phe_GUG) was separated into the 5′-side primer fragment (pP) and the 3′-side template fragment (T) shown in red and gray, respectively. The altered bases of T3 are indicated in blue. (B) A primer/template assay for simultaneously measuring the kinetics of adenylylation and G₋₁ addition reactions by Thg1 with pP1-T1. Representative single-turnover assays with pP1-T1 for determining k_obs for adenylylation in the presence of ATP and for G₋₁ addition in the presence of both ATP and GTP. The reactions shown are time courses of activity with 10 µM CaThg1 in excess over pP1-T1; aliquots from each time point were treated with phosphatase (CIP), followed by resolution on urea-PAGE gel. RNA fragments were detected by SybrGold staining. (C) Time course experiments of G₋₁ addition reaction with D-stem loop variants of two-piece tRNA; pP1-T1 (♦), pP2-T1 (▴), pP3-T1 (•), pP4-T1 (▪). Lines represent each time course fitted to a single-exponential equation (Equation 1) to yield k_obs. (D) Time course experiments of G₋₁ addition reaction by using short primer (pP4) and template fragment variants (T1, T2, and T3); pP4-T1 (▪), pP4-T2 (♦), pP4-T3 (•). The bars in the graphs are SD of more than two independent experiments.