Insertion suppressor mutations are mutations that alter the tRNA's anticodon loop by expanding the anticodon. Tetracodon-anticodon interactions are more stable than corresponding tricodon-anticodon interactions, suggesting that at higher temperatures, the probably rare tetra-anticodons are advantaged as compared to tri-anticodons and contribute more to translation. These expanded tetra-anticodons match quadruplet codons, or tetracodons.
In silico predictions of mitochondrial tetracoded polypeptides translated from regular mitochondrial protein coding genes frequently align with protein sequences existing in GenBank. Comparing complete mitochondrial genomes from different species, analyses show that predicted numbers of tetra-anticodons (from antisense tRNAs) coevolve with predicted numbers of tetracoded genes. This is also observed for expanded putative anticodons in tRNA sidearm loops, suggesting that these sidearm loops also contribute, by unknown mechanism(s), to translation.
Mitochondrial tetracodon numbers predicted by alignment methods increase with mean lizard body temperature, as expected by greater codon-anticodon stabilities, indicating that tetracoding is an adaptation for translation at high temperatures.
In addition, deamination gradient analyses show that predicted overlapping tetragenes follow less deamination gradients than other regions of the same genes, as expected for overlap coding regions less free to follow spontaneous chemical mutations because constrained by two coding frames, rather than only one. Codon usages in these regions include fewer circular code codons (circular code codons enable reading frame retrieval) than surrounding, non-overlap coding regions, as expected by the circular code theory for reading frame retrieval.
These analyses indicate that additional protein coding genes are coded by genetic codes with expanded codons. The possibility of pentacodons forming pentagenes should also be explored in the future. This body of results awaits direct experimental confirmations.