Transplantation of a tyrosine editing domain into a tyrosyl-tRNA synthetase variant enhances its specificity for a tyrosine analog

Abstract

To guarantee specific tRNA and amino acid pairing, several aminoacyl-tRNA synthetases correct aminoacylation errors by deacylating or "editing" misaminoacylated tRNA. A previously developed variant of Escherichia coli tyrosyl-tRNA synthetase (iodoTyrRS) esterifies or "charges" tRNA(Tyr) with a nonnatural amino acid, 3-iodo-l-tyrosine, and with l-tyrosine less efficiently. In the present study, the editing domain of phenylalanyl-tRNA synthetase (PheRS) was transplanted into iodoTyrRS to edit tyrosyl-tRNA(Tyr) and thereby improve the overall specificity for 3-iodo-l-tyrosine. The beta-subunit fragments of the PheRSs from Pyrococcus horikoshii and two bacteria were tested for editing activity. The isolated B3/4 editing domain of the archaeal PheRS, which was exogenously added to the tyrosylation reaction with iodoTyrRS, efficiently reduced the production of tyrosyl-tRNA(Tyr). In addition, the transplantation of this domain into iodoTyrRS at the N terminus prevented tyrosyl-tRNA(Tyr) production most strongly among the tested fragments. We next transplanted this archaeal B3/4 editing domain into iodoTyrRS at several internal positions. Transplantation into the connective polypeptide in the Rossmann-fold domain generated a variant that efficiently charges tRNA(Tyr) with 3-iodo-l-tyrosine, but hardly produces tyrosyl-tRNA(Tyr). This variant, iodoTyrRS-ed, was used, together with an amber suppressor derived from tRNA(Tyr), in a wheat germ cell-free translation system and incorporated 3-iodo-l-tyrosine, but not l-tyrosine, in response to the amber codon. Thus, the editing-domain transplantation achieved unambiguous pairing between the tRNA and the nonnatural amino acid in an expanded genetic code.

Fig. 1.

Isolation of editing domains. (A) The domains of the bacterial and archaeal/eukaryotic PheRS β-subunits and the analyzed fragments. (B–D) Acidic PAGE analysis of tyrosyl-tRNA^Tyr formation in the presence of iodoTyrRS and the E. coli (B), T. thermophilus (C), and P. horikoshii (D) fragments. The bands corresponding to tyrosyl-tRNA^Tyr and uncharged tRNA^Tyr are marked. The reactions for lanes 2–6 contained iodoTyrRS, whereas those for lanes 3–6 additionally contained the PheRS fragment indicated above each lane. Neither iodoTyrRS nor any fragment was included in the reaction for lanes 1.

Fig. 2.

Transplantation of editing domains into iodoTyrRS. (A) The domains of iodoTyrRS and its variants. Numbers indicate the first residue of each domain. A filled symbol represents each enzyme, and the same symbol is used to indicate the data for the enzyme in B–G. (B and D) Time courses of tyrosyl-tRNA^Tyr formation in the presence of iodoTyrRS, the indicated fusion/insertion variants, the indicated editing-deficient fusion variants, and no enzyme. (C and E) Formation of tyrosyl-tRNA^Tyr (lanes Y) and iodotyrosyl-tRNA^Tyr (lanes IY) in the presence of the indicated enzymes. The bands corresponding to aminoacyl-tRNA^Tyr and uncharged tRNA^Tyr are marked. (F) Time courses of tyrosyl-tRNA^Tyr hydrolysis in the presence of N-Ped-IYRS, CP-Ped-IYRS, and no enzyme. (G) The translation of GST(Am) in the wheat germ cell-free system. The indicated enzymes were added to the reactions, together with the suppressor tRNA, in the absence (−IY) and presence (IY) of 3-iodo-l-tyrosine. All of the reactions contained l-tyrosine. The produced full-length GST was detected by Western blotting.

Fig. 3.

Comparison among the tertiary structures of editing aaRSs. (Left) Structural model of a CP-Ped-IYRS dimer complexed with a tRNA^Tyr. The model was built as described in SI Text. (Center and Right) The crystal structures of the E. coli ThrRS·tRNA^Thr complex (Center) (14) and the P. horikoshii LeuRS·tRNA^Leu complex (Right) (10) are shown for comparison. The two monomers of CP-Ped-IYRS are shown in blue and cyan with their editing domains in magenta and red, respectively. The editing domain is in red, with the remaining part of the enzyme in cyan, for ThrRS and LeuRS. tRNA is in lime green in each structure. The distances between the aminoacylation and editing active sites are shown.