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 β-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.