Fig 2: Lack of Thr-to-Ala mistranslation despite effective mischarging of Ala to tRNAThr(G4:U69). (A) Both Thr-AGU(G4:U69) and Thr-CGU(G4:U69), but not Thr-AGU(U4:G69), can be mischarged with Ala by AlaRS but not ThrRS. The reaction was carried out with 1 μM AlaRS or ThrRS and 20 μM tRNAThr; AlaRS alone without tRNA was used as negative control. (B) Chimeric tRNAThr with G3:U70 (made by switching the original C3:G70 and G4:U69 base pairs) can be mischarged with Ala by AlaRS with enhanced efficiency. The reaction was carried out with 200 nM AlaRS and 4 μM Ala-AGC or Thr-AGU(G3:U70) tRNA, or 20 μM Thr-AGU(G4:U69) tRNA. (C) No Thr-to-Ala substitution was observed in either HEK293 cell lysate or enriched GAPDH from HEK293 cells even under various indicated manipulations to enhance Ala mischarging. OE: overexpression; N.D.: not detected.

Fig 3: Cross-editing of Ala-tRNAThr by ThrRS. (A) Human ThrRS could efficiently deacylate Ala-tRNAThr. Different concentrations of ThrRS as indicated were incubated with 2 μM 3H-labeled Ala-tRNAThr. In the following assays, 200 nM enzyme (ThrRS or AlaRS) and 2 μM Ala-tRNAThr were used in the reaction unless specified. (B) AlaRS harboring an editing domain similar to that of ThrRS cannot deacylate Ala-tRNAThr. (C) Residues in ThrRS involved in deacylation of Ser-tRNAThr are important for editing Ala-tRNAThr. Top: alanine-3′-aminoadenosine, an analog of Ala-tRNAThr terminus, was modeled into editing site of E. coli ThrRS based on the structure of E.coli ThrRS in complex with serine-3′-aminoadenosine (PDB ID: 1TKY). The equivalent residue numbers in human ThrRS are shown in parenthesis. The methyl group of threonine is modeled and shown as a dashed circle, which would clash with Asp259 and His159. Bottom: Both D259A and H155A/H159A mutants completely abolish the editing activity of human ThrRS towards Ala-tRNAThr. (D) Editing-defective ThrRS H155A/H159A mutant (ThrRS-Mut) can mischarge serine, but not alanine, onto tRNAThr. (E) ThrRS can deacylate Ala-tRNAThr in the presence of AlaRS. ThrRS-Mut may slightly enhance the release of Ala-tRNAThr from AlaRS and thus the turnover of AlaRS. The reaction was carried out with 1 μM AlaRS and 20 μM tRNAThr, with or without 500 nM ThrRS/ThrRS-Mut. (F) E. coli ThrRS is able to deacylate human Ala-tRNAThr with similar efficiency as human ThrRS.