Role of tRNA orthogonality in an expanded genetic code

Abstract

We found that Methanocaldococcus jannaschii DSM2661 tyrosyl-tRNA synthetase (Mj E9RS), specifically evolved to charge its cognate tRNA with the unnatural amino acid p-acetylphenylalanine (pAcF) in E. coli, misaminoacylates the endogenous E. coli prolyl-tRNAs with pAcF at a low level (0.5% per proline frequency) in both the absence or presence of its co-evolved amber suppressor tRNA (M. jannaschii tyrosyl-tRNA, tRNA_CUA^MjTyr). In contrast to other E. coli tRNAs, the identity elements for recognition of the proly tRNAs by the E. coli prolyl-tRNA synthetase (C1, G72, and A73) are similar to those in tRNA_CUA^MjTyr. Although the unique acceptor stem identity elements of the prolyl-tRNAs likely lower their recognition by the other endogenous aaRSs in E. coli, resulting in enhanced fidelity in the wild type strain, they lead to misaminoacylation by the archae-derived E9RS. Misincorporation of pAcF for proline was resolved to below detectable levels by overexpression of the endogenous E. coli prolyl-tRNA synthetase (proS) gene in combination with additional genomic manipulations to further increase the intracellular ratio of the ProS over its cognate proline tRNAs. These experiments suggest another mechanism by which the cell maintains the high fidelity of protein biosynthesis.