Modulation of translational decoding by m⁶A modification of mRNA

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.