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. 2021 Sep 29;12:5706. doi: 10.1038/s41467-021-25948-y

Fig. 1. Discovery and quantification of selection derived qtRNAs.

Fig. 1

a Schematic representation of cellular reporter assays. Suppressed positions in the reporter proteins are shown in parentheses. In all cases, reporter protein translation prematurely terminates in the absence of a functional qtRNA due to a stop codon generated downstream of the quadruplet codon. Conversely, a functional qtRNA affects quadruplet codon suppression, yielding a full-length protein. b Validation of the luciferase reporter for quadruplet codon suppression. The previously described qtRNAThrACCA25 (n = 12 biologically independent samples) selectively decodes the cognate quadruplet codon, whereas a codon/anticodon mismatch or native triplet tRNA yields no luminescence using the quadruplet codon at residue S357 in LuxAB. Data for the remaining combinations represents the mean and standard deviation of three biologically independent samples. c Decoding efficiency of previously described qtRNAs, qtRNAGlnCAAA27, and qtRNAGlyGGGG85 (n = 8 biologically independent samples). d The LacZ selection pipeline yielded functional qtRNAs for aspartate (n = 4 biologically independent samples), glutamate (Glu n = 8 except for Glu-AGGG n = 7), histidine (His n = 4 except for His-AGGA n = 3 and His-UAGA n = 6), and tyrosine (Tyr n = 7 except for Tyr-CCCC, CCCU, CCAU, and UAGA n = 8 and Tyr-CUGC, CCAG, and UACA n = 4), which were assayed using the luciferase reporter. e Several LacZ selection-derived qtRNAs showed robust suppression of the cognate quadruplet codon at the permissive residue Y151 in sfGFP (n = 8 biologically independent samples, except for Tyr-UAGA n = 7). f The sfGFP reporter was used to quantify amino acid incorporation at position Y151 via mass spectrometry. Comprehensive peptide fragmentation spectra are reported in Supplementary Fig. 3 and summary LC-MS/MS results are reported in Supplementary Table 8. In all cases, reporter data is normalized to an otherwise wild-type reporter protein and color-coded by the used reporter. Unless otherwise noted, all qtRNAs were assayed against their cognate quadruplet codon incorporated at residue S357 in LuxAB or Y151 in sfGFP. Data represent the mean and standard deviation as appropriate.