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.

转移 RNA (tRNA) 的反密码子修饰可微调核糖体上的密码子识别，以实现准确翻译。细菌和古细菌分别利用 tRNA ^Ile反密码子中修饰的胞苷、赖氨酸 (L) 和胍丁胺 (agm ² C)来破译 AUA 密码子。 L 和 agm ² C 含有带有极性末端的长侧链，但它们的功能仍然难以捉摸。在这里，我们报告了识别核糖体上 AUA 密码子的 tRNA ^Ile的低温电子显微镜结构。两种修饰均通过独特的 C-A 几何结构与密码子的第三个腺嘌呤相互作用。侧链向mRNA的3'方向延伸，极性末端与AUA密码子3'端残基的2'-OH形成氢键。生化分析表明，修饰胞苷的极性末端与第四个 mRNA 残基的 2'-OH 之间的额外相互作用促进了 AUA 解码。我们还可视化了另一种 tRNA 修饰环状N ⁶ -苏氨甲酰基腺苷 (ct ⁶ A)，并揭示了 ct ⁶ A 如何有助于高效解码的分子基础。