The frequency of errors upon decoding of messenger RNA by the bacterial ribosome is low, with one misreading event per 1 × 10⁴ codons. In the universal genetic code, the AUN codon box specifies two amino acids, isoleucine and methionine. In bacteria and archaea, decoding specificity of the AUA and AUG codons relies on the wobble avoidance strategy that requires modification of C34 in the anticodon loop of isoleucine transfer RNA^Ile_CAU (tRNA^Ile_CAU). Bacterial tRNA^Ile_CAU with 2-lysylcytidine (lysidine) at the wobble position deciphers AUA while avoiding AUG. Here we report cryo-electron microscopy structures of the Escherichia coli 70S ribosome complexed with elongation factor thermo unstable (EF-Tu) and isoleucine-tRNA^Ile_LAU in the process of decoding AUA and AUG. Lysidine in tRNA^Ile_LAU excludes AUG by promoting the formation of an unusual Hoogsteen purine–pyrimidine nucleobase geometry at the third position of the codon, weakening the interactions with the mRNA and destabilizing the EF-Tu ternary complex. Our findings elucidate the molecular mechanism by which tRNA^Ile_LAU specifically decodes AUA over AUG.

细菌核糖体解码信使RNA时的错误频率较低，每1 × 10 ⁴密码子就有1个误读事件。在通用遗传密码中，AUN 密码子盒指定了两种氨基酸：异亮氨酸和蛋氨酸。在细菌和古细菌中，AUA 和 AUG 密码子的解码特异性依赖于摆动避免策略，该策略需要修改异亮氨酸转移 RNA ^Ile _CAU (tRNA ^Ile _CAU )反密码子环中的 C34 。摆动位置带有 2-赖氨酰胞苷（赖氨酸）的细菌 tRNA ^Ile _{CAU破译 AUA，同时避免 AUG。在这里，我们报道了}大肠杆菌70S 核糖体在解码 AUA 和 AUG 过程中与热不稳定延伸因子 (EF-Tu) 和异亮氨酸-tRNA ^Ile _LAU复合的冷冻电镜结构。 tRNA ^Ile _LAU中的赖氨酸通过促进密码子第三个位置上不寻常的 Hoogsteen 嘌呤-嘧啶核碱基几何形状的形成、削弱与 mRNA 的相互作用并破坏 EF-Tu 三元复合物的稳定性来排除 AUG。我们的研究结果阐明了 tRNA ^Ile _LAU特异性解码 AUA 而非 AUG 的分子机制。