Structural insights into the decoding capability of isoleucine tRNAs with lysidine and agmatidine

The anticodon modifications of transfer RNAs (tRNAs) finetune the codon recognition on the ribosome for accurate translation. Bacteria and archaea utilize the modified cytidines, lysidine (L) and agmatidine (agm²C), respectively, in the anticodon of tRNA^Ile to decipher AUA codon. L and agm²C contain long side chains with polar termini, but their functions remain elusive. Here we report the cryogenic electron microscopy structures of tRNAs^Ile recognizing the AUA codon on the ribosome. Both modifications interact with the third adenine of the codon via a unique C-A geometry. The side chains extend toward 3' direction of the mRNA, and the polar termini form hydrogen bonds with 2'-OH of the residue 3'-adjacent to the AUA codon. Biochemical analyses demonstrated that AUA decoding is facilitated by the additional interaction between the polar termini of the modified cytidines and 2'-OH of the fourth mRNA residue. We also visualized cyclic N⁶-threonylcarbamoyladenosine (ct⁶A), another tRNA modification, and revealed a molecular basis how ct⁶A contributes to efficient decoding.