RESUMO
Heterologous tRNA:aminoacyl tRNA synthetase pairs are often employed for noncanonical amino acid incorporation in the quest for an expanded genetic code. In this work, we investigated one possible mechanism by which directed evolution can improve orthogonal behavior for a suite of Methanocaldococcus jannaschii ( Mj) tRNATyr-derived amber suppressor tRNAs. Northern blotting demonstrated that reduced expression of heterologous tRNA variants correlated with improved orthogonality. We suspected that reduced expression likely minimized nonorthogonal interactions with host cell machinery. Despite the known abundance of post-transcriptional modifications in tRNAs across all domains of life, few studies have investigated how host enzymes may affect behavior of heterologous tRNAs. Therefore, we measured tRNA orthogonality using a fluorescent reporter assay in several modification-deficient strains, demonstrating that heterologous tRNAs with high expression are strongly affected by some native E. coli RNA-modifying enzymes, whereas low abundance evolved heterologous tRNAs are less affected by these same enzymes. We employed mass spectrometry to map ms2i6A37 and Ψ39 in the anticodon arm of two high abundance tRNAs (Nap1 and tRNAOptCUA), which provides (to our knowledge) the first direct evidence that MiaA and TruA post-transcriptionally modify evolved heterologous amber suppressor tRNAs. Changes in total tRNA modification profiles were observed by mass spectrometry in cells hosting these and other evolved suppressor tRNAs, suggesting that the demonstrated interactions with host enzymes might disturb native tRNA modification networks. Together, these results suggest that heterologous tRNAs engineered for specialized amber suppression can evolve highly efficient suppression capacity within the native post-transcriptional modification landscape of host RNA processing machinery.