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. 2020 Apr 8;48(11):6170–6183. doi: 10.1093/nar/gkaa221

Figure 4.

Figure 4.

G at position 34 of the anticodon is prone to miscoding in eukaryotes. (A) The heat map represents, in various eukaryotic genomes, the ratio between the number of genes encoding any of the 64 anticodon combinations and the total number of tRNA genes. The colour code of the heat map is shown on the right, from black for highly represented genes to white for tRNA genes that are absent in genomes. Arrows indicate the suppressor tRNA genes containing anticodons corresponding to the stop codons and tRNA genes containing the anticodons 34GGN36 and 34GAG36. (B) Human tRNA transcripts tRNAAla,Pro,Thr,Ser,Leu containing 34ANN36 anticodons and their impossible tRNA (itRNA) counterparts, which contains respectively a single substitution of A at position 34 of the anticodons to G, are introduced into HeLa cells. The histogram represents the Relative Metabolic Activity (RMA) average value measured by WST1 assay for each itRNA transcript (in red) normalized to the corresponding Wt transcript (in orange) (n = 9, Statistical Student Test, ns: P-value > 0.05, *: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001, ****: P ≤ 0.0001). The error bars represent the standard deviation for each value. (C) Human tRNAAla transcripts containing the major determinant 3G-U70 for Alanyl-tRNA synthetase with variable anticodon sequences are introduced in Rabbit Reticulocyte Lysates together with a synthetic HA-tagged Renilla reporter mRNA that contains silent mutations at Phenylalanine, Proline, Threonine, Valine, Leucine and Serine codons in order to enrich the proportions of following codons Phenylalanine (UUC), Proline (CCC), Threonine (ACU, ACC), Valine (GUC), Leucine (CUC), Serine (UCC, UCU) according to Supplementary Figure S9. (D) The yield of Renilla proteins synthesized in the presence of tRNAAla transcripts of without transcript (Ø) is evaluated by SDS-PAGE of 35S-methionine-labeled proteins. (E) Histogram representing the relative luciferase activities of the synthesized Renilla proteins normalized to the luciferase activity obtained in absence of tRNA transcript (Ø) (n = 3, ns: P-value>0.05, **: P ≤ 0.01).