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).

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.

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).