Fig. 2: Engineering RF2 to attenuate UGA termination.
From: Engineering a genomically recoded organism with one stop codon
a, Left, comparative mRNA transcript and protein diagrams displaying key nucleotides and amino acids in wild-type RF2.B (cyan) and mutant RF2.B3 (purple). Right, RF2 variants used in this study. b, Top, AlphaFold48 structural prediction of RF2.B3 with residue deviations from wild-type RF2 marked in purple. Bottom, magnified views of wild-type RF2 SPF (left) and mutant RF2 PPF (right) codon recognition loops, highlighting interactions with residue 205. AlphaFold48 structural prediction suggests that wild-type S205 presents up to four hydrogen bonds within the RF2 codon recognition loop, whereas P205 presents one. c, Schematic representation of mCherry–YFP fluorescent reporter used to assess release activity at three target codons (X is UAG, UGA or UAA) relative to a sense codon control (GCG) in a peptide linker between mCherry and YFP. Release activity at codon X prevents downstream YFP expression and fluorescence without compromising mCherry, whereas stalling and degradation leads to loss of mCherry and YFP signals. High YFP fluorescence suggests readthrough. d, Schematic representation of expected fluorescent outcomes resulting from the mCherry–YFP readthrough assay. e,f, mCherry (top) and YFP fluorescence (bottom) expressed as a function of translation termination or readthrough at target codon X during reporter translation in variant strains with release factors. Fluorescence is normalized to GCG codon construct fluorescence in absence (e) or presence (f) of UAG- or UGA-suppressing supD tRNAs. Colour scheme in d–f as indicated in c. a.u., arbitrary units. Data are mean ± s.e.m. P values for comparison with rEc∆1.∆A by unpaired t-tests (n = 3 biological replicates). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
