Abstract

The order in which the amino acids were added to the genetic code was previously inferred from consensus among forty metrics. Many of these reflect abiotic abundance on ancient Earth. However, the abundances that matter are those within primitive cells that already had sophisticated RNA and perhaps peptide metabolism. Here, we directly infer the order of recruitment from the relative ancestral amino acid frequencies of ancient protein sequences. Small size predicts ancient amino acid enrichment better than the previous consensus metric does. We place metal-binding and sulfur-containing amino acids earlier than previously thought, highlighting the importance of metal-dependent catalysis and sulfur metabolism to ancient life. Understanding early life has implications for our search for life elsewhere in the universe.