Frequent occurrence and predicted functions of tRNAs with 4-base-pair anticodon stems in bacteria: extended superwobble hypothesis.

Recently, a tRNATrpCCA with a 4-base-pair (bp) anticodon stem (AS) was shown to efficiently recognize a near-cognate UGA codon in unicellular eukaryotes, such as some trypanosomatids and ciliates, thereby representing a novel codon reassignment mechanism. To determine whether this mechanism also evolved in bacteria, we analysed a dataset of 42 109 genomes, including previously reported cases of stop-to-tryptophan UGA reassignment and a newly identified instance in the phylum Patescibacteriota. We show that the 4-bp AS tRNATrp species are present across diverse bacteria and in some cases likely function in decoding in-frame UGA codons. Most notable is the endosymbiotic bacterium Candidatus Zinderia insecticola, which contains only the near-cognate 4-bp AS tRNATrpCCA, while lacking both canonical 5-bp AS tRNATrpCCA and a tRNATrpUCA. The secondary structure of this 4-bp AS tRNATrp resembles that of its eukaryotic counterpart, suggesting convergent evolution. We experimentally confirmed the UGA readthrough capacity of 4-bp AS tRNATrpCCA in Escherichia coli, and applied molecular dynamics simulations to suggest the underlying mechanism. Furthermore, we tested several predictions based on accepting the previously excluded possibility of C:A base pairing at the 3rd codon position. These findings provide new insights into the structural diversity of transfer RNAs (tRNAs) and expand our understanding of genetic code evolution.