Order of amino acid recruitment into the genetic code resolved by Last Universal Common Ancestor’s protein domains

The current “consensus” order in which amino acids were added to the genetic code is based on potentially biased criteria such as absence of sulfur-containing amino acids from the Urey-Miller experiment in which no sulfur was added. Here, we reassess this order by exploiting the fact that proteins that emerged prior to the genetic code’s completion are likely enriched in early amino acids and depleted in late amino acids. We identify the most ancient protein-coding sequences born prior to the archaeal-bacterial split. Amino acid usage in protein sequences whose ancestors date back to a single homolog in the Last Universal Common Ancestor (LUCA) largely matches the consensus order. However, our findings indicate that metal-binding (cysteine and histidine) and sulfur-containing (cysteine and methionine) amino acids were added to the genetic code much earlier than previously thought. Surprisingly, even more ancient protein sequences — those that had already diversified into multiple distinct copies in LUCA — show a different pattern to single copy LUCA sequences: significantly less depleted in the late amino acids tryptophan and tyrosine, and enriched rather than depleted in phenylalanine. This is compatible with at least some of these sequences predating the current genetic code. Their distinct enrichment patterns thus provide hints about earlier, alternative genetic codes.