Glutamine codon-driven translational readthrough reveals context-dependent stop codon decoding fidelity

Deviations from the canonical genetic code include reassignment of UAA/UAG stop codons to glutamine in divergent eukaryotes, and tRNA^Gln has been shown to mediate near-cognate stop codon readthrough in canonical-code organisms. However, the sequence determinants and mechanistic basis of this decoding event remain poorly understood. Using ribosome profiling, quantitative immunoblotting, and mass spectrometry in Saccharomyces cerevisiae, we demonstrate that premature stop codon readthrough efficiency is governed by both local glutamine codon context and the global glutamine codon content of the mRNA. A QXQ motif flanking the stop codon promotes baseline readthrough, which is amplified in proportion to total transcript glutamine codon abundance. Mass spectrometry confirms that glutamine is specifically inserted at the premature stop, with no flanking miscoding, implicating tRNA^Gln competition with the release factor as the mechanistic basis of readthrough. Consistent with this model, yeast proteins terminating in short C-terminal glutamine repeats are evolutionarily enriched for strong stop codon contexts, suggesting selective pressure to reinforce termination fidelity at readthrough-prone loci.