There are two important issues in Kachale et al. 2023 to resolve from the outset, both clarified in Supporting Information. One: a reported 5 bp AS tryptophan tRNA (in their Fig. 3a) which did not lead to efficient stop codon readthrough7, likely originates from a bacterium present in the ciliate culture, not from Condylostoma magnum. Thus, Condylostoma probably only has nuclear genome-encoded 4 bp AS tRNATrpCCA’s, further supporting the proposal that they are necessary for efficient UGA translation as tryptophan. Two: the authors express uncertainty about the genetic codes used by several ciliate species, particularly relating to UGA codons and tRNAs with potential complementary anticodons; this can partly be traced to incorrect annotations in reference databases or earlier publications, that have since been superseded. This is pertinent to the interpretation of eRF1 substitutions and their role in UGA translation.

With their diverse genetic codes, ciliates are ideal for exploring hypotheses about these codes. Consistent with Kachale et al’s 4 bp AS tryptophan tRNA proposal for translation of UGA as tryptophan, we find such tRNAs occur not only in Condylostoma magnum (class Heterotrichea; UAR=Q/*, UGA=W/*)2,3 but also in another ciliate species with an ambiguous genetic code, Loxodes magnus (class Karyorelictea; UAG=Q, UAA=Q/*? [UAA may also be a stop], UGA=W/*)8, and in the ciliate genus Blepharisma (class Heterotrichea; Fig. 1), which appears to translate UGA unambiguously as tryptophan (UAR=*, UGA=W)8. A draft Loxodes magnus somatic genome assembly has over one hundred 4 bp AS tRNATrp genes but just six 5 bp AS tRNATrp genes (Fig. 1). The 5 bp AS tRNAs of L. magnus shown in Fig 1. have one or two unpaired T-stem bases and are co-located on a contig with similar sequences and secondary structures, particularly the T-stem, but different anticodons (CUU, CUA, CCU) corresponding to non-tryptophan codons. This suggests relaxation of selection, and that these are likely pseudogenes. In contrast, two other heterotrich ciliates with standard genetic codes9,10, Stentor coeruleus and Fabrea salina, only have 5 bp AS tRNATrp genes (Fig. 1). Among members of the Paramecium aurelia complex, which are not known to translate UGA as tryptophan (code: UAR=Q, UGA=*)2, we found a ciliate species, Paramecium biaurelia, with at least one 4 bp AS tRNA (Extended Data Fig. 1a) among its eight tRNATrp genes. For such tRNAs and additional ones in other organisms that are not evident pseudogenes, the ability to translate UGA as tryptophan should be carefully experimentally investigated in future.

Predicted tryptophan tRNAs encoded in the macronuclear genomes of heterotrich and karyorelict ciliates.

The two ciliate classes are indicated by different background colours: Heterotrichea - green; Karyorelictea - cyan. Nucleotide substitutions that differ between tRNA genes are indicated by double arrows. Stop codon reassignments are given under the species names. See Extended Data Fig. 1 for Blepharisma’s tRNAUCA predictions (selenocysteine and mitochondrial). Blepharisma japonicum and Blepharisma undulans tRNATrpCCA also have 4 bp anticodon stems (Source Data Fig. 1 and Extended Data Fig. 1).

In a human pathogenic trypanosomatid Leishmania species, tRNA C-to-U editing of the wobble (5’) anticodon base of nuclear genome-encoded mitochondrial tRNATrpCCA’s generates UCA anticodons that enable mitochondrial UGA codon translation11. Kachale et al. reported no editing of cytosolic Blastocrithidia tRNATrpCCA’s7 that could permit translation of cytosolic UGAs. Previously, tRNA sequencing did not reveal C-to-U editing of the Condylostoma magnum tRNATrpCCA anticodon wobble base that would generate an anticodon complementary to UGA codons2. We have also observed no appreciable C-to-U editing (> 0.1% of tryptophan tRNAs) in Blepharisma and Loxodes 4 bp AS tRNATrp sequences (in 172,929 and 9,721 unique reads, respectively; Supplementary Table 1, Source Data Fig. 1).

Anticipating the discovery of natural 4 bp AS tRNATrpCCA’s in Blastocrithidia and the ciliates Condylostoma, Blepharisma, and Loxodes, the idea that tRNA AS mutations can enhance decoding of near-cognate codons was previously explored in back-to-back papers by Schultz and Yarus12,13. In the first paper, by extensive mutational screening of Escherichia coli tRNA su7 G36, a derivative of a tryptophan suppressor tRNA with a CUG anticodon, they found that mutations that disrupt the top AS stem base pair — creating a 4 bp AS stem — led to the most translation of UAG12, which involves G-U wobble pairing at the 1st codon position that is normally disallowed. In the second paper, using su7 tRNACUA they showed that mutations the disrupt the top AS stem base pair promote UAA translation13, which involves mismatched 3rd position A-C like that required for UGA translation by 4bp AS tRNACCA’s7.

Particular amino acid substitutions in homologs of the protein that recognizes stop codons, eRF1, were formerly thought to be associated with loss of stop codon recognition necessary for the evolution of particular genetic codes in ciliates14,15. With the benefit of additional eRF1 sequences and ciliate genetic codes, we previously reported multiple counterexamples to such associations2. Kachale et al. proposed that a single amino acid substitution in eRF1, from Ser67 to Ala/Gly67 (numbered with respect to yeast eRF1), may be needed for loss of UGA termination in conjunction with a shorter tRNATrp anticodon stem for efficient UGA translation as tryptophan7. However, this substitution is present in eRF1’s of multiple ciliates that use the standard genetic code: Stentor coeruleus, Fabrea salina and Climacostomum virens (all members of class Heterotrichea; like Condylostoma and Blepharisma; Fig. 2a). eRF1 of the ciliate Pseudocohnilembus persalinus (class Oligohymenophorea), which has the genetic code UAR=Q and UGA=*, also has Ala67, and so too does eRF1 of the diplomonad flagellate Giardia intestinalis (standard genetic code).

eRF1 substitutions and potential signals of eRF1-tRNA competition in Blepharisma.

(a) eRF1 coordinates are given according to that of Saccharomyces cerevisiae, and the alignment window is the same as that in Kachale et al. 2023. The complete eRF1 alignment along with the sources of the sequences is provided in Source Data Fig. 2. For the genetic codes of each species, stars indicate stop codons, and question marks indicate possible stop codons. Check marks and crosses respectively indicate agreements and disagreements with respect to the proposed UGA assignment/eRF1 substitution at position 67. The eRF1 phylogeny to the left was generated by RAxML. The second Geleia acuta eRF1 paralog is encoded by an incomplete transcript. (b) Codon frequency upstream of predicted B. stoltei stop codons for the permutations of “T”, “G” and “A” bases. For the complete codon frequency matrix, see Extended Data Fig. 2a.

Furthermore, all karyorelict ciliates translate UGA as tryptophan within mRNA coding sequences (and use it as a stop at the ends of coding sequences)8. One species, Loxodes magnus, has eRF1’s with Ala67, but other species’ eRF1’s have either Ser67 or Cys67 (Fig. 2a). Recently the ciliate species Plagiopyla frontata (class Plagiopylea) was reported to have ambiguous UGA codons that are translated as tryptophan in coding sequences (genetic code UAR=Q, UGA=W/*)1. eRF1 from this distantly related ciliate has Ser67. So too does eRF1 from the alveolate Amoebophrya sp. ex Karlodinium venificum. Thus, the Ala/Gly67 substitution is present in ciliate species without UGA translation and is not necessary in multiple ciliate species which translate UGA as tryptophan. Ala/Gly67 also appears unnecessary in the Amoebophrya species.

Unlike bacteria which have two proteins that recognize two stop codons each, RF1 and RF2, standard genetic code model eukaryotes, like yeast, typically have a single “omnipotent” protein, eRF1, that recognizes all three stop codons. eRF1 paralogs were previously noted to have arisen independently in certain ciliate genera, including Euplotes and Tetrahymena16,17,18. The frequent occurrence of such paralogs in ciliates raises the possibility some may have subfunctionalized, like RF1 and RF2, with different stop codon recognition capabilities, but this needs experimental determination. Blepharisma stoltei has three divergent eRF1 paralogs (60-72% amino acid identity for the three pairwise comparisons), of which the most highly transcribed one (BSTOLATCC_MAC3627; mean 540 RPKM, standard deviation 50 RPKM for a developmental time series19) has Ala67, but there is also an eRF1 paralog with low transcription (mean 9.9 RPKM, standard deviation 6.2 RPKM) and Ser67 (Fig. 2a). Gene expression of the more divergent Blepharisma eRF1 paralogs is comparable to that of the ancient eRF1 paralog Dom34/Pelota (BSTOLATCC_MAC12938; mean 6.8 RPKM; standard deviation 2.3 RPKM), a protein responsible for the translation-associated process “No-Go decay”16. It is conceivable that these paralogs have functionally diverged, now serving an alternative role like Dom34/Pelota.

Though we have only observed UGA codons translated as tryptophan in Blepharisma stoltei, in vitro translation experiments suggest Blepharisma japonicum’s ortholog of the highly transcribed B. stoltei eRF1, also with Ala67, can recognize all three stop codons, but UGA the most weakly20. Correspondingly, with some capacity of Blepharisma eRF1 to recognize UGA as stop codons, a signal of potential competition between B. stoltei eRF1 and tRNATrp can be observed in the form of UGA codon depletion in a region beginning 25-30 codons upstream of UAR stop codons (Fig. 2b). A similar depletion was observed in the karyorelict ciliates and the heterotrich ciliate Condylostoma which do use UGA as a stop close to transcript ends2,8. Interestingly, depletion of UAA and UGA codons occurs in a similar region before stops in Blastocrithidia nonstop, contrasting with the constancy of reassigned UAG codons7. This suggests eRF1-tRNA competition not only for UAA but also for UGA in this species.

While the type of amino acid substitution proposed by Kachale et al. may certainly substantially enhance translation, it should be noted that such substitutions are not a prerequisite for the acquisition of a new genetic code under the hypothesis which best fits the evolution of the ambiguous stop/sense genetic codes, the “ambiguous intermediate hypothesis”21. Instead, in a transitional evolutionary phase, codons may be interpreted in two ways, with potential eRF1-tRNA competition. With time, beneficial mutations or modifications in either the tRNA or eRF1 (or other components of translation) that reduce competition may be selected.

Instead of focusing on individual eRF1 substitutions, we propose future investigations should more generally explore the structure of non-standard genetic code eRF1’s captured in translation termination in the context of their own ribosomes. New genetic codes involving stop codon reassignments have had ample opportunity to evolve in eukaryotes through a combination of tRNA and eRF1 mutations, but are limited to just a few clades, most notably having radiated in ciliates. We thus infer that an additional aspect has enabled genetic code evolution in these prolific microbes, and continue to suggest that this may be their ability to either tolerate or resolve genetic code ambiguity.


Conceptualization: E.C.S. Investigation: E.C.S., B.K.B.S, M.S. Methodology: E.C.S, C.E., B.K.B.S, A.S. Resources: all authors. Writing: all authors. Supervision: E.C.S.

Competing interests

The authors declare no competing interests.

Figure captions

Secondary structures of Paramecium biaurelia 4 bp AS tRNATrpCCA and Blepharisma tRNAUCA’s and a tRNA-like molecule with a possible UCA anticodon.

(a) Paramecium biaurelia tRNATrpCCA; terminal nucleotides predicted by tRNAscan-SE 2.0. (b) B. stoltei tRNASecUCA. (c) B. stoltei mitochondrial tRNATrpUCA. (d) tRNA-like molecule with potential UCA anticodon in B. stoltei. (e) Multiple sequence alignment of tRNA-like sequences from Blepharisma spp. (f) Multiple sequence alignment of paralogs of tRNA-like sequence paralogs in B. stoltei ATCC30299. (g) YAMAT-seq mapping to the B. stoltei ATCC30299 mitochondrial genome.

Codon usage before stops and base frequencies around stops.

(a) Codon frequency upstream of predicted B. stoltei stop codons. (b) Base frequencies flanking B. stoltei TGA codons (n=44087). (c) Adenosine frequency of +4 at base immediately downstream of translated B. stoltei codons. (d) Base frequencies around TAA (n=21002) stop codons. (e) Base frequencies around TAG (n=4707) stop codons. (f) Base frequencies around TGG tryptophan codons (n=78535).