PMC

Single-turnover progress curves for α-phosphorothioate-substituted adenylate and 2′- and 3′-substituted variants at tRNA A76 using ThrRS wild-type enzyme. (A) R_p (filled squares) and S_p (filled circles) α-phosphorothioate-substituted ATP analogs. (B) Modified A76 tRNAs (2′-dA76-, 3′-dA76-, and tRNA^Thr-periodate-treated). The 3′-dA76 (open squares) and tRNA^Thr-periodate treated (filled triangles) served as the controls for the reaction.

(A) X-ray crystal structure of ribosomal substrates bound in the active site [PDB entry 1VQN (78)]. The α-NH₃ group of the A-site substrate has been replaced with a hydroxyl group to inhibit the reaction. The A76 2'-OH hydrogen bonds with the α-NH₃, and the 2'- and 3'-OHs coordinate a solvent molecule on their far side. (B) Schematic of the peptidyl transfer intermediate and the proposed mechanism for resolution into products. The 2'-OH acts as a proton shuttle by simultaneously donating a proton to the 3'-OH and accepting a proton from the α-NH₃. (C) X-ray crystal structure of the transition state mimic CCA-phosphate-puromycin_Phe-CC [PDB entry 1VQP (78)]. The non-bridging phosphate oxygens mimic the peptide and the oxyanion. The proposed proton transfers are indicated with arrows.

The mechanism of aminoacyl transfer mediated by the 2′-OH of tRNA A76 in E. coli ThrRS. Dotted lines represent H-bonds, and the arrows indicate the proton relay. See Discussion for the detailed explanation of the mechanism.

The role of the 3′-OH of A76 in editing. (A and B) Tyrosylation of A76 tRNA^Phe (squares), 3′-dA76 tRNA^Phe (triangles), and 3′-F-A76 tRNA^Phe (circles) by 1 μM wild-type PheRS (A) or 1 μM βG318W PheRS (B). (C) Deacylation of 0.2–0.4 μM tyrosylated A76 tRNA^Phe, 3′-dA76 tRNA^Phe, and 3′-F-A76 tRNA^Phe in the presence (filled symbols) or absence (open symbols) of 0.5 μM wild-type β-subunit PheRS.