Resurrecting tRNAs of the Last Universal Common Ancestor (Commonote) Toward Rebuilding the Ancient Translation System

Ancestral sequence reconstruction (ASR) was used to study the evolution of the translational system by inferring tRNA sequences of the Last Archaeal Common Ancestor (LACA), the Last Bacterial Common Ancestor (LBCA), and the Last Universal Common Ancestor (LUCA), referred to as "Commonote" in our previous studies. A composite phylogenetic tree inferred from the reconstructed ancestral tRNA sequences revealed a topology distinct from those of previously inferred aminoacyl-tRNA synthetase (ARS) trees. This incongruence may reflect changes in tRNA structural features or distinct evolutionary pressures on tRNAs and ARSs, suggesting a more nuanced evolutionary history of the translation system, but it may also arise from methodological differences between studies. Phylogenetic analysis identified conserved identity elements, such as the discriminator base and anticodon, across tRNAs for different amino acids, supporting their status as ancestral features. Most LUCA tRNA species retain bacterial identity elements, potentially enabling their use in modern systems such as Escherichia coli, although exceptions, including tRNA^Gly and initiator tRNA^Met, may present functional limitations. To evaluate the translational functionality of LUCA tRNAs and ancient codon-anticodon interactions, synthesized LUCA tRNA^Ala and tRNA^Ser variants with anticodons decoding ACN codons were tested in an E. coli reconstituted in vitro translation system. These reconstructed tRNAs successfully decoded ACN codons, with tRNA^Ala variants exhibiting translation efficiencies comparable to those of native E. coli tRNA^Thr. Notably, decoding patterns differed from Crick's and extended wobble rules, as anticodons with various first-position bases recognized multiple third codon positions. This study offers new insights into the early evolution of the translation system and demonstrates the functional potential of ancestral tRNAs in contemporary contexts.