N⁶-methyladenosine (m⁶A) is an abundant, dynamic mRNA modification that regulates key steps of cellular mRNA metabolism. m⁶A in the mRNA coding regions inhibits translation elongation. Here, we show how m⁶A modulates decoding in the bacterial translation system using a combination of rapid kinetics, smFRET and single-particle cryo-EM. We show that, while the modification does not impair the initial binding of aminoacyl-tRNA to the ribosome, in the presence of m⁶A fewer ribosomes complete the decoding process due to the lower stability of the complexes and enhanced tRNA drop-off. The mRNA codon adopts a π-stacked codon conformation that is remodeled upon aminoacyl-tRNA binding. m⁶A does not exclude canonical codon-anticodon geometry, but favors alternative more dynamic conformations that are rejected by the ribosome. These results highlight how modifications outside the Watson-Crick edge can still interfere with codon-anticodon base pairing and complex recognition by the ribosome, thereby modulating the translational efficiency of modified mRNAs.

N ⁶ -甲基腺苷 (m ⁶ A) 是一种丰富的动态 mRNA 修饰，可调节细胞 mRNA 代谢的关键步骤。mRNA 编码区中的m ^{6 A 抑制翻译延伸。}在这里，我们展示了 m ⁶ A 如何结合快速动力学、smFRET 和单粒子冷冻电镜来调节细菌翻译系统中的解码。我们表明，虽然修饰不会损害氨酰基-tRNA 与核糖体的初始结合，但在 m ⁶ A 存在的情况下，由于复合物稳定性较低和 tRNA 脱落增强，完成解码过程的核糖体较少。mRNA 密码子采用 π 堆叠密码子构象，在氨酰基-tRNA 结合后进行重构。m ⁶ A 并不排除规范的密码子-反密码子几何结构，但倾向于被核糖体拒绝的替代性更动态的构象。这些结果强调了 Watson-Crick 边缘之外的修饰仍然可以干扰密码子-反密码子碱基配对和核糖体的复杂识别，从而调节修饰的 mRNA 的翻译效率。