Large-scale transfer RNA analysis suggests an ancestral code

The model predicts a traceable consequence. If modern tRNAs descended from those hairpins, they might retain copies of those triplets in the same region, a paired section called the acceptor stem. Triplets are groups of three RNA letters.

Conserved signals persist across many lineages and through evolutionary time. The researchers looked for conserved triplets in the region where the mini-helix model predicts they should survive.

After filtering nearly half a million sequences, the team analyzed more than 70,000 tRNA genes from nearly 5,000 genomes across bacteria, archaea and eukaryotes. The researchers describe the resulting data set as the largest used to date for this type of analysis.

Could RNA folding alone explain the triplets?

The team compared actual sequences with simulated versions of equivalent structure. That control tested whether folding and other structural constraints could explain the patterns without an older matching system.

In several groups of tRNAs, triplets appeared more frequently than the simulations predicted. The excess frequency suggests that structure alone may not explain every signal. It does not prove the evolutionary interpretation, but it makes the proposed ancestry more plausible.

Ribas de Pouplana said: “The key was to verify that the signal persisted when taking into account the structural constraints of the molecule. This control supports the interpretation that the observed patterns may contain information about their evolutionary history.”

In plain terms, the observed patterns go beyond what RNA shape alone would predict.

Does the signal match the code’s expansion?

The researchers compared signal strength with a proposed timeline for adding amino acids to the genetic code. In general, the patterns followed that chronology:

• TRNAs associated with glycine, alanine and valine showed stronger signals. The proposed timeline places those amino acids earlier in the code.
• TRNAs associated with tyrosine and tryptophan showed weaker signals. The timeline places those amino acids later.

This agreement provides additional support for the proposed progressive development of the genetic code. Because the comparison uses a proposed chronology, it offers supporting evidence rather than a direct recording of the process.

In plain terms, the signal was strongest for amino acids proposed to enter the code earlier.

What remains unresolved?

The team also detected triplets in other arms of the tRNA molecule. The mini-helix hypothesis does not explain that observation on its own. The exact pathway from early RNA structures to present-day molecules therefore remains open.

The paper credits two undergraduate students as first authors. Both completed placements in IRB Barcelona’s Gene Translation Laboratory. The article, “Molecular fossils of an ancestral code revealed by large-scale analysis of tRNAs,” was published by Ash Nova Royo et al. in PNAS in 2026. Its DOI is 10.1073/pnas.2627541123.

For researchers testing origin models, the practical gain is a genome-wide comparison that controls for RNA structure; the evolutionary route remains unsettled.