Codon degeneracy contributes to divergent fitness effects of rare tRNAs with A-starting anticodons

Transfer RNA (tRNA) repertoires vary greatly across genomes, shaped by genetic drift and selection. A peculiar pattern across prokaryotes is the near-complete absence of tRNAs with unmodified adenine at the 34th (wobble) position (i.e., tRNAANN). Each of these tRNAs are just a single mutation away from several other tRNAs. Hence, their persistent absence suggests fundamental but hitherto unclear constraints. We engineered 36 Escherichia coli strains expressing tRNAs carrying each theoretically possible ANN anticodon to determine their functionality and fitness effects. Notably, there was no evidence of broad toxicity due to these tRNAs. All five tRNAANN tested underwent post-transcriptional maturation and all seven tested compensated for the deletion of their respective native tRNABNN (carrying G, C or U at the 34th position), demonstrating that tRNAANN are translationally active. Furthermore, tRNAANN from four-fold degenerate (4D) codon boxes were unmodified and were generally neutral or beneficial, whereas tRNAANN from two-fold degenerate (2D) boxes underwent A34-to-I34 modification and were more likely to impair fitness. We suggest superwobbling by tRNAANN -- decoding an entire four-codon set -- as one mechanism underlying these differential fitness effects. Maximal degeneracy in 4D boxes buffers or exploits tRNAANN superwobbling via synonymous decoding, whereas constrained degeneracy in 2D boxes renders it deleterious, likely through amino acid misincorporation. Thus, these differential fitness effects, sharpens the paradox of neutral or beneficial yet absent 4D tRNAANN, while beginning to empirically unravel underlying causes for the absence of 2D tRNAANN.