Fig. 4: Codon competition and sense codon reassignment of the four-fold degenerate valine codon box.

a Reported readthrough of each valine codon by t7tRNAs²². b MALDI-MS analysis of the pre-charged tRNA mixtures reveals the stoichiometric balance of the mixtures, which is shown in the pie chart. c Heatmap displaying the results of the codon competition experiments, where darker green corresponds with higher readthrough and the corresponding readthrough value numerically shown inside the box. The expected codon readthrough, based on patterns revealed by surveying the leucine codons, is depicted with a black box. A table of observed vs expected MS values is shown in SI Table 4. d Representative mass spectra of two codon competition experiments: one for the GUA codon and one for the GUC codon. All other mass spectra are shown in SI Fig. 5. e A single mRNA with three valine codons is decoded in the PURE translation system supplemented with two pre-charged valine tRNA isoacceptors, resulting in the translation of a peptide with valine and tert-butyl glycine (tBu-Gly highlighted in green). S12 ribosomes and t7tRNA were paired for these translation experiments, which were performed in duplicate. f MALDI-MS analysis of the translation results (expected mass of 2023.99 Da). Source data are provided as a Source Data file.