Evolutionary tuning impacts the design of bacterial tRNAs for the incorporation of unnatural amino acids by ribosomes.

In order to function on the ribosome with uniform rate and adequate accuracy, each bacterial tRNA has evolved to have a characteristic sequence and set of modifications that compensate for the differing physical properties of its esterified amino acid and its codon-anticodon interaction. The sequence of the T-stem of each tRNA compensates for the differential effect of the esterified amino acid on the binding and release of EF-Tu during decoding. The sequence and modifications in the anticodon loop and core of tRNA impact the codon-anticodon strength and the ability of the tRNA to bend during codon recognition. These discoveries impact the design of tRNAs for the efficient and accurate incorporation of unnatural amino acids into proteins using bacterial translation systems.