Fig. 5: Characterization of AUA decoding by ava2C34 with synthetic mRNAs.
From: A tRNA modification with aminovaleramide facilitates AUA decoding in protein synthesis
a, Schematic depiction of tRNA binding to the A-site of the E. coli ribosome using synthetic mRNAs with atomic substitutions at the fourth nucleotide (N)—unmodified (2′-OH, left), 2′-H (second), 2′-O-methyl nucleoside (2′-OMe, third) and 2′-F (right). The P-site was occupied by E. coli tRNAGlu, followed by incubation with 32P-labeled P. putida tRNAIle2 to examine AUA decoding upon 2′-OH substitutions at the fourth residue. b, Ribosome-binding ability of P. putida tRNAIle2 on the AUA codon of mRNAs with different 2′-OH substitutions at the fourth residue, namely, unmodified (2′-OH), 2′-H, 2′-OMe and 2′-F. Nonspecific tRNA binding was estimated without (w/o) mRNA. The binding ratio was calculated as the ratio of bound tRNA to input tRNA. Data are shown as mean ± s.d. (n = 5 technical replicates). Tukey’s adjusted P values are as follows: P = 0.002 (2′-OH versus 2′-H), P = 0.0048 (2′-OH versus 2′-OMe) and P < 0.0001 (2′-H versus 2′-F and 2′-OMe versus 2′-F). *P < 0.05. c–f, Comparison of base pairing geometry and side chain orientation of ava2C34 upon binding to the AUA codon with different 2′-OH substitutions at the fourth residue—A4 (2′-OH, c), dA4 (2′-H, d), A(F)4 (2′-F, e) and Am4 (2′-OMe, f). On interacting with Am4 mRNA, the ava2C side chain represents the branched density, to which models A (green) and B (yellow) are assigned.
