Fig. 3: The nucleotide sequence downstream the premature stop codon’s effect on the probability of stop codon readthrough.

A The effect of the identity of the nucleotides in a 5-amino acid window upstream of the stop codon on stop codon readthrough (SCR) probability. Statistical analysis was performed using Pearson correlation and two-sided testing. B High G content in a 5-amino acid window downstream of the stop codon does not increase the probability of SCR, while T decreases it. Statistical analysis was performed using Pearson correlation and two-sided testing. C Reporters were binned into three categories based on the SCR rates, and nucleotide frequencies were calculated 1-nt upstream and downstream of the stop codons. The identity of the base immediately before the stop codon did not impact SCR probability. The identity of the base immediately after the stop codon impacted SCR probability. T increases, and G decreases the protein synthesis termination efficiency. D Stop codon readthrough events occurred primarily due to amino acid misincorporations identified by mass spectrometry. TGA, in blue, was almost always replaced by tryptophan. TAA, in purple, was mainly replaced by alanine, glutamine, and tryptophan, while TGA, in green, was replaced by glutamine and tyrosine. Misincorporation of several other amino acids at the stop codon was clearly a minor process (relative abundance <10%). These MS experiments were performed with the reporters after purification with His-tag affinity resin. Thus, we could not quantify the likelihood of SCR events. However, the MS analyses did provide information on the relative abundance of the amino acids’ misincorporation, presented in Supplementary Table 4. E The downstream mutation to T and the upstream mutation to ATTAT reduced the likelihood of SCR events for all three tested positions. To quantify the SCR likelihood, we measured the His-tag expression with western blot in E. coli cells transformed with these mutants, grown at 18 °C. We calculated the percentage of His-tag expression compared with the internal positive control (PC, wild-type mScarlet). Source data are provided as a Source data file.