Sense-to-sense changes in bacterial genetic codes: Experimental validation by cell-free translation and native-host proteomics

In the sixty years since the publication of the complete genetic code [1], many variants of the classical codon table [2] have been observed to occur naturally, including both stop codon and sense-to-sense reassignment, but in bacteria the only experimentally validated variants are stop-codon reassignments. Recently, computational analysis with Codetta predicted bacterial genetic codes containing sense-to-sense changes. Using an Escherichia coli cell-free protein synthesis (CFPS) system, we tested tRNAs predicted to reassign arginine codons to tryptophan. These candidate CGG-decoding tRNAs from bacteria phylogenetically distant from E. coli restored cyan fluorescence from an sfGFP Y66R reporter in a dose-dependent manner when added to CFPS extracts containing canonical tRNAArg(CCG). Reactions lacking the CGG-to-Trp candidate tRNA remained near background. Reassignment was also observed with firefly luciferase and NanoLuc reporters, in which Trp incorporation restored activity and solubility. Intact-protein mass spectrometry yielded masses consistent with Arg-to-Trp substitution. Deep data-independent acquisition (DIA) proteomics of Anaerococcus prevotii, a species for which Codetta predicts CGG to code for Trp, revealed six peptides containing Trp encoded at native CGG codons and no peptides containing CGG-encoded Arg, consistent with native CGG decoding as Trp in this organism. Together, these results provide direct functional support for predicted bacterial sense-to-sense genetic codes and establish a cell-free framework for discovering natural tRNAs that expand translational coding capacity.