Abstract
Summary The updated Wobble Hypothesis reasonably explains why some 40 tRNA species are sufficient to decode the 61 amino acid codons of the Universal Genetic Code. However, we still have no clue why eubacteria lack tRNA isoacceptors with ANN anticodons, whereas eukaryotes universally lack eight GNN anticodons, only one of which is also absent in bacteria. Direct tRNA sequencing could resolve the patterns of nucleoside modification that had been driving the divergent evolution in prokaryotes and eukaryotes, but this task will require the development of AI‐supported base‐callers that can recognize modified nucleosides without any subsequent analytical verification. Our knowledge of the bacterial tRNA landscape is moreover broadened by the recent discovery of antisense tRNAs and tRNA‐derived fragments that should be examined in their roles for gene expression, translation, bacterial physiology or metabolism.