title: 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.
authors.affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Oki
authors.forename: Kensaku
authors.initials: K
authors.lastname: Sakamoto
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Hiroshi M
authors.initials: HM
authors.lastname: Sasaki
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama