An RNA modification prevents extended codon-anticodon interactions from facilitating +1 frameshifting.

RNA post-transcriptional modifications act by stabilizing the functional conformations of RNA. While their role in messenger RNA (mRNA) decoding is well established, it is less clear how transfer RNA (tRNA) modifications outside the anticodon contribute to tRNA stability and accurate protein synthesis. Absence of such modifications causes translation errors, including mRNA frameshifting. By integrating single-molecule fluorescence resonance energy transfer and cryogenic electron microscopy, we demonstrate that the N-methylguanosine (mG) modification at position 37 of Escherichia coli tRNA is necessary and sufficient for modulating the conformational energy of this tRNA on the ribosome so as to suppress +1 frameshifting otherwise induced by this tRNA. Six structures of E. coli ribosomal complexes carrying tRNA lacking mG37 show this tRNA forms four and even five codon-anticodon base pairs as it moves into the +1 frame, allowing direct visualization of the long-standing hypothesis that a four base pair codon-anticodon can form during +1 frameshifting.