Codon degeneracy contributes to divergent fitness effects of rare tRNAs with A34

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.