Investigations of ciliate UGA translation

Ciliates, a widespread clade of eukaryotic microbes, have the most known nuclear genetic codes (eight), with the latest addition from their best-studied class (Oligohymenophorea). All alternative ciliate genetic codes involve stop codon reassignments. Two of these codes are ambiguous, with “stop” codons either translated or terminating translation, depending on their context. Ambiguous genetic codes may have originated independently three times in ciliates, and twice more in trypanosomatids from the genus Blastocrithidia and an alveolate species from the genus Amoebophrya. What all these codes share is translation of UGA “stop” codons as tryptophan. For the 20 standard amino acids, though tRNA genes with complementary anticodons to UAA and UAG have invariably been found in ciliate species in which these codons are translated, vexingly, this has not been so for species with UGA tryptophan codons. Furthermore, a UGA-cognate cysteine tRNA(UCA) was reported in Euplotes, the only ciliate genus that translates UGA as cysteine. Recently, Kachale et al. hypothesised UGA translation in Blastocrithidia and the ciliate Condylostoma magnum may be due to a 4 base pair anticodon stem (AS) in tryptophan tRNAs, rather than the usual 5 base pair stems. New tRNA data from additional ciliates bolsters this hypothesis. Kachale et al. also hypothesised a particular amino acid substitution in the key stop codon recognition protein, eRF1 (eukaryotic Release Factor 1), enhances UGA translation as tryptophan. However, we report multiple independent instances of the proposed substitution that have not led to UGA translation, and multiple ciliate species with UGA tryptophan translation but without the substitution, indicating it is not necessary. Consistent with the ambiguous intermediates hypothesis for genetic code evolution, experimental evidence and our observations suggest continued ciliate eRF1-tRNA competition at codons both molecules may recognize. Contrary to Kachale et al., to evolve new genetic codes via stop codon reassignment, we instead propose substitutions favouring reduced competition by eRF1 do not need to occur concomitantly with tRNA alterations.