Modulation of translational decoding by m⁶A modification of mRNA

N-6-methyladenosine (m(6)A) is an abundant, dynamic mRNA modification that regulates key steps of cellular mRNA metabolism. m(6)A in the mRNA coding regions inhibits translation elongation. Here, we show how m(6)A 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 m(6)A fewer ribosomes complete the decoding process due to the lower stability of the complexes and enhanced tRNA drop-off. The mRNA codon adopts a & pi;-stacked codon conformation that is remodeled upon aminoacyl-tRNA binding. m(6)A 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. m(6)A is an mRNA modification that slows down translation elongation. Here, Jain et al. show that m(6)A delays decoding and increases tRNA drop-off from the ribosome by favoring alternative codon conformations that are rejected by the ribosome.