A general approach for the generation of orthogonal tRNAs.
BACKGROUND
The addition of new amino acids to the genetic code of Escherichia coli requires an orthogonal suppressor tRNA that is uniquely acylated with a desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. A tRNA(Tyr)(CUA)-tyrosyl-tRNA synthetase pair imported from Methanococcus jannaschii can be used to generate such a pair. In vivo selections have been developed for selecting mutant suppressor tRNAs with enhanced orthogonality, which can be used to site-specifically incorporate unnatural amino acids into proteins in E. coli.


RESULTS
A library of amber suppressor tRNAs derived from M. jannaschii tRNA(Tyr) was generated. tRNA(Tyr)(CUA)s that are substrates for endogenous E. coli aminoacyl-tRNA synthetases were deleted from the pool by a negative selection based on suppression of amber nonsense mutations in the barnase gene. The remaining tRNA(Tyr)(CUA)s were then selected for their ability to suppress amber nonsense codons in the beta-lactamase gene in the presence of the cognate M. jannaschii tyrosyl-tRNA synthetase (TyrRS). Four mutant suppressor tRNAs were selected that are poorer substrates for E. coli synthetases than M. jannaschii tRNA(Tyr)(CUA), but still can be charged efficiently by M. jannaschii TyrRS.


CONCLUSIONS
The mutant suppressor tRNA(Tyr)(CUA) together with the M. jannaschii TyrRS is an excellent orthogonal tRNA-synthetase pair for the in vivo incorporation of unnatural amino acids into proteins. This general approach may be expanded to generate additional orthogonal tRNA-synthetase pairs as well as probe the interactions between tRNAs and their cognate synthetases.
Wang
L
L
Department of Chemistry, University of California at Berkeley, 94720, USA.
Schultz
P G
PG
D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D005816:Genetic Complementation Test; D017017:Methanococcus / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012343:RNA, Transfer / Q000138:chemical synthesis* / Q000235:genetics; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013489:Suppression, Genetic / Q000235:genetics
D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases