A tRNA modification with aminovaleramide facilitates AUA decoding in protein synthesis

Modified tRNA anticodons are critical for proper mRNA translation during protein synthesis. It is generally thought that almost all bacterial tRNAs^Ile use a modified cytidine-lysidine (L)-at the first position (34) of the anticodon to decipher the AUA codon as isoleucine (Ile). Here we report that tRNAs^Ile from plant organelles and a subset of bacteria contain a new cytidine derivative, designated 2-aminovaleramididine (ava²C). Like L34, ava²C34 governs both Ile-charging ability and AUA decoding. Cryo-electron microscopy structural analyses revealed molecular details of codon recognition by ava²C34 with a specific interaction between its terminal amide group and an mRNA residue 3'-adjacent to the AUA codon. These findings reveal the evolutionary variation of an essential tRNA modification and demonstrate the molecular basis of AUA decoding mediated by a unique tRNA modification.