Criteria for (A) LUCA Pfam annotation, (B) identifying HGT to be filtered, and (C) pre-LUCA Pfam annotation. Details are in Methods, with a brief summary here. (A) Pruning HGT between archaea and bacteria reveals a LUCA node as dividing bacteria and archaea at the root. Colored circles are indicated just upstream of the most recent common ancestor (MRCA) of all copies of that Pfam found within the same taxonomic supergroup. We recognize a total of five bacterial supergroups [FCB, PVC, CPR, Terrabacteria, and Proteobacteria (25, 26)] and four archaeal supergroups [TACK, DPANN, Asgard, and Euryarchaeota (27, 28)]; only 4 out of 5 bacterial supergroups and 3 out of 4 archaeal supergroups are shown. The yellow diamond indicates LUCA as a speciation event between archaea and bacteria. We do not assume that the LUCA coalescence timing was the same for every Pfam (29). Prior to HGT pruning, PVC sequences can be found on either side of the two lineages divided by the root. After pruning intradomain HGT, four MRCAs are found one node away from the root, and three more MRCAs are found two nodes away from the root, fulfilling our other LUCA criterion described in the Methods, namely the presence of at least three bacterial and at least two archaeal supergroup MRCAs one to two nodes away from the root. (B) Criteria for pruning likely HGT between archaea and bacteria (see Materials and Methods for details). We partition into monophyletic groups of sequences in the same supergroup; in this example, there are four such groups, representing two bacterial supergroups and one archaeal supergroup. There is one “mixed” node, separating an archaeal group (HG1) from a bacterial group (HG2). It is also annotated by GeneRax (19) as a transfer “T.” The bacterial nature of groups 3 and 4 indicates a putative HGT direction from group 2 to group 1. Group 2 does not contain any Euryarchaeota sequences, meeting the third and final requirement for pruning of group 1. If neither Proteobacteria nor Euryarchaeota sequences were present among the other descendants of the parent node, both groups 1 and 2 would be considered acceptors of a transferred Pfam and would both be pruned from the tree. (C) Pre-LUCA Pfams have at least two nodes annotated as LUCA. — PNAS via PubMed
The current “consensus” order in which amino acids were added to the genetic code is based on potentially biased criteria, such as the absence of sulfur-containing amino acids from the Urey-Miller experiment which lacked sulfur. More broadly, abiotic abundance might not reflect biotic abundance in the organisms in which the genetic code evolved.
Here, we instead identify which protein domains date to the last universal common ancestor (LUCA) and then infer the order of recruitment from deviations of their ancestrally reconstructed amino acid frequencies from the still-ancient post-LUCA controls.
We find that smaller amino acids were added to the code earlier, with no additional predictive power in the previous consensus order. Metal-binding (cysteine and histidine) and sulfur-containing (cysteine and methionine) amino acids were added to the genetic code much earlier than previously thought.
Methionine and histidine were added to the code earlier than expected from their molecular weights and glutamine later. Early methionine availability is compatible with inferred early use of S-adenosylmethionine and early histidine with its purine-like structure and the demand for metal binding.
Even more ancient protein sequences-those that had already diversified into multiple distinct copies prior to LUCA-have significantly higher frequencies of aromatic amino acids (tryptophan, tyrosine, phenylalanine, and histidine) and lower frequencies of valine and glutamic acid than single-copy LUCA sequences.
If at least some of these sequences predate the current code, then their distinct enrichment patterns provide hints about earlier, alternative genetic codes.
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