Abstract:

<p>Modification of anticodon nucleotides allows tRNAs to decode multiple codons, expanding the genetic code. Additionally, modifications located in the anticodon loop but outside the anticodon itself, stabilize tRNA-codon interactions, increasing decoding fidelity. Anticodon loop nucleotide 37 is 3&#39; to the anticodon and in tRNA , is methylated at the N1 position in its nucleobase (mG37). The mG37 modification in tRNA stabilizes its interaction with the codon and maintains the &nbsp;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 tRNA mG37 modification is important for the association step during binding to a cognate CCG codon. In contrast, mG37 prevented association to a slippery CCC-U or +1 codon. Similar analyses of frameshift suppressor tRNASufA6, a tRNA derivative containing an extra nucleotide in its anticodon loop that undergoes +1 frameshifting, revealed that mG37 destabilizes interactions with both the cognate CCG and slippery codons. One reason for this destabilization was the disruption of a conserved U32&middot;A38 nucleotide pairing in the anticodon stem through insertion of G37.5. Restoring the tRNA U32&middot;A37.5 pairing resulted 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&middot;A37.5 at 3.6-Å resolution showed 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.</p>