Importance of a tRNA anticodon loop modification and a conserved, noncanonical anticodon stem pairing in for decoding.

Modification of anticodon nucleotides allows tRNAs to decode multiple codons, expanding the genetic code. Additionally, modifications located in the anticodon loop, outside the anticodon itself, stabilize tRNA–codon interactions, increasing decoding fidelity. Anticodon loop nucleotide 37 is 3′ to the anticodon and, in , is methylated at the N1 position in its nucleobase (mG37). The mG37 modification in stabilizes its interaction with the codon and maintains the mRNA frame. However, it is unclear how mG37 affects binding at the decoding center to both cognate and +1 slippery codons. Here, we show that the mG37 modification is important for the association step during binding to a cognate CCG codon. In contrast, mG37 prevented association with a slippery CCC-U or +1 codon. Similar analyses of frameshift suppressor tRNA, a derivative containing an extra nucleotide in its anticodon loop that undergoes +1 frameshifting, reveal that mG37 destabilizes interactions with both the cognate CCG and slippery codons. One reason for this destabilization is the disruption of a conserved U32·A38 nucleotide pairing in the anticodon stem through insertion of G37.5. Restoring the tRNA U32·A37.5 pairing results in a high-affinity association on the slippery CCC-U codon. Further, an X-ray crystal structure of the 70S ribosome bound to tRNA U32·A37.5 at 3.6 Å resolution shows a reordering of the anticodon loop consistent with the findings from the high-affinity measurements. Our results reveal how the tRNA modification at nucleotide 37 stabilizes interactions with the mRNA codon to preserve the mRNA frame.