Authors
A Kachale1; Z Pavlíková2; A Nenarokova1; A Roithová2; I Durante1; P Miletínová2; K Záhonová1; S Nenarokov1; J Votýpka3; E Horáková1; RL Ross4; V Yurchenko5; P Beznosková2; LS Valášek2; Z Paris1; J Lukeš1; 1 Institute of Parasitology, Biology Centre, Czech Academy of Sciences, České Budějovice, (Budweis), Czech Republic; 2 Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic; 3 Faculty of Science, Charles University, BIOCEV, Prague, Czech Republic; 4 Thermo Fisher Scientific, Franklin, MA, United States; 5 Life Science Research Centre, Faculty of Science, University of Ostrava, Ostrava, Czech Republic Discussion
Transfer RNA (tRNA) delivers a specific amino acid residue to ribosomes. Its anticodon pairs with the complementary mRNA codon according to the universal genetic code, which defines translation termination by three stop codons. Remarkably, some protists have reassigned all three stop codons to sense codons, neglecting this fundamental principle. Performing an across- the-genome analysis of in-frame stops in 7,259 predicted protein-coding genes of the newly isolated trypanosomatid Blastocrithidia nonstop we show that their distribution is not random and their representation diminishes with increasing protein abundance. Robust comparisons with other trypanosomatids revealed specific features at the end of the coding regions defining UAA as the only functional termination codon. We demonstrate that while novel tRNAsGlu fully cognate to UAG and UAA evolved to reassign these two stops, recoding of UGA followed an unprecedented path via shortening the anticodon stem-loop of tRNATrpCCA from 5 to 4 base pairs (bp). While tRNATrp with a canonical 5-bp long stem-loop recognizes UGG as dictated by the genetic code, its shortened version efficiently incorporates tryptophan also into in-frame UGA to allow translation to continue. Mimicking this radical evolutionary twist by engineering and overexpressing both stem loop variants of tRNATrpCCA from B. nonstop, Trypanosoma brucei and Saccharomyces cerevisiae in the latter two species, we recorded a significantly higher readthrough for all 4-bp stem-loop variants. The phenomenon is specific for tRNATrp, since decoding by other two S. cerevisiae tRNAs near-cognate to UGA (tRNACys and tRNAArg) was unaffected by alterations in their stem-loop length. Furthermore, we demonstrate that a specific mutation in the release factor 1 (eRF1) of B. nonstop specifically restricts UGA recognition and thus robustly potentiates the UGA recoding to tryptophan. Revealing that the same strategy has also been independently adopted by the ciliate Condylostoma magnum, we propose that these two key alterations co-evolved synergistically. Altogether, we have defined a novel and universal mechanism underlying the stop codon recognition by specific variants of tRNAsTrp in combination with mutated eRF1, which has been exploited in unrelated eukaryotes with reassigned stop codons.
