tRNA anticodon stem length variations are critical for stop codon reassignment

Published on by Anna Nenarokova
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 basis of the genetic code. 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 unexpectedly defining UAA as the only functional termination codon. Strikingly, we demonstrate that while novel Glu-tRNAs fully cognate to UAG and UAA evolved to reassign these two stops, recoding of UGA followed an unprecedented path by 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 in addition efficiently incorporates tryptophan to in-frame UGAs 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 read-through 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 conclude that 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.

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Czech Science Foundation grant (GACR) 20-07186S

Czech Science Foundation grant (GACR) 18-15962S

Czech Science Foundation grant (GACR) 20-11585S

Czech Science Foundation grant (GACR) 20-00579S

ERC CZ grant LL1601

Czech Ministry of Education 16_0000759

Czech Bioimaging grant LM2018129

Praemium Academiae grant provided by the Czech Academy of Sciences to Leos Shivaya Valasek

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