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Transplantation of a tyrosine editing domain into a tyrosyl-tRNA synthetase variant enhances its specificity for a tyrosine analog.
Transplantation of a tyrosine editing domain into a tyrosyl-tRNA synthetase variant enhances its specificity for a tyrosine analog.
- Source :
-
Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2008 Sep 09; Vol. 105 (36), pp. 13298-303. Date of Electronic Publication: 2008 Sep 02. - Publication Year :
- 2008
-
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.
- Subjects :
- Amino Acid Motifs
Cell-Free System
Escherichia coli genetics
Escherichia coli metabolism
Models, Molecular
Protein Biosynthesis
Protein Engineering
Protein Structure, Tertiary
Pyrococcus horikoshii genetics
Pyrococcus horikoshii metabolism
Substrate Specificity
Thermus thermophilus genetics
Thermus thermophilus metabolism
Tyrosine-tRNA Ligase genetics
Tyrosine analogs & derivatives
Tyrosine metabolism
Tyrosine-tRNA Ligase chemistry
Tyrosine-tRNA Ligase metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1091-6490
- Volume :
- 105
- Issue :
- 36
- Database :
- MEDLINE
- Journal :
- Proceedings of the National Academy of Sciences of the United States of America
- Publication Type :
- Academic Journal
- Accession number :
- 18765802
- Full Text :
- https://doi.org/10.1073/pnas.0803531105