A general approach for the generation of orthogonal tRNAs

作者：Wang L; Schultz PG^*

来源：Chemistry & Biology, 2001, 8(9): 883-890.

DOI：10.1016/S1074-5521(01)00063-1

• aminoacyl-tRNA synthetase
• in vivo selection
• in vivo unnatural amino acid mutagenesis
• orthogonal suppressor tRNA

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(CUA)(Tyr)-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 AY. jannaschii tRNA(Tyr) was generated. tRNA(CUA)(Tyr) 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 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 tRNAC(CUA)(Tyr) but still can be charged efficiently by M. jannaschii TyrRS.
Conclusions: The mutant suppressor tRNA(CUA)(Tyr) 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.

• 出版日期2001-9