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

Candidate CGG-decoding tRNAs from distant bacteria restore Trp incorporation in an Escherichia coli cell-free system, enabling Arg-to-Trp substitution at native CGG codons.

• Why it matters: Bacterial genetic-code variants beyond stop-codon reassignment have lacked experimental validation, leaving predicted sense-to-sense changes unconfirmed.
• What they did: Using Escherichia coli CFPS with sfGFP Y66R, luciferase, and NanoLuc reporters, they tested Codetta-predicted CGG-to-Trp tRNAs and confirmed Arg-to-Trp masses by intact-protein MS.
• The result: DIA proteomics of Anaerococcus prevotii found six Trp-containing peptides at native CGG codons and no CGG-encoded Arg peptides, supporting native CGG decoding as Trp.

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