Short tRNA anticodon stem and mutant eRF1 allow stop codon reassignment

• Published in: Nature (London) Vol. 613; no. 7945; pp. 751 - 758
• Main Authors: Kachale, Ambar, Pavlíková, Zuzana, Nenarokova, Anna, Roithová, Adriana, Durante, Ignacio M., Miletínová, Petra, Záhonová, Kristína, Nenarokov, Serafim, Votýpka, Jan, Horáková, Eva, Ross, Robert L., Yurchenko, Vyacheslav, Beznosková, Petra, Paris, Zdeněk, Valášek, Leoš Shivaya, Lukeš, Julius
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 26.01.2023; Nature Publishing Group
• Subjects: 14/63; 38/43; 38/70; 38/71; 45/91; 631/326/417/2548; 631/337/574/1793; 82/58; 82/80; Amino acids; Anticodon - chemistry; Anticodon - genetics; Anticodon - metabolism; Ciliophora - genetics; Codon, Terminator - genetics; Codons; Eukaryotes; Eukaryotic Cells; Genes; Genetic code; Genetic Code - genetics; Genomes; Humanities and Social Sciences; multidisciplinary; Mutation; Peptide Termination Factors - genetics; Peptide Termination Factors - metabolism; Proteins; Ribosomes; RNA, Transfer - genetics; RNA, Transfer - metabolism; RNA, Transfer, Glu - genetics; RNA, Transfer, Trp - genetics; Saccharomyces cerevisiae - genetics; Science; Science (multidisciplinary); Stems; Stop codon; Transfer RNA; tRNA; Trypanosoma brucei brucei - genetics; Tryptophan
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/s41586-022-05584-2

Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists have reassigned all stop codons as sense codons, neglecting this fundamental principle 1 – 4 . Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop . We reveal that in this species in-frame stop codons are underrepresented in genes expressed at high levels and that UAA serves as the only termination codon. Whereas new tRNAs Glu fully cognate to UAG and UAA evolved to reassign these stop codons, the UGA reassignment followed a different path through shortening the anticodon stem of tRNA Trp CCA from five to four base pairs (bp). The canonical 5-bp tRNA Trp recognizes UGG as dictated by the genetic code, whereas its shortened 4-bp variant incorporates tryptophan also into in-frame UGA. Mimicking this evolutionary twist by engineering both variants from B. nonstop , Trypanosoma brucei and Saccharomyces cerevisiae and expressing them in the last two species, we recorded a significantly higher readthrough for all 4-bp variants. Furthermore, a gene encoding B. nonstop release factor 1 acquired a mutation that specifically restricts UGA recognition, robustly potentiating the UGA reassignment. Virtually the same strategy has been adopted by the ciliate Condylostoma magnum . Hence, we describe a previously unknown, universal mechanism that has been exploited in unrelated eukaryotes with reassigned stop codons. Analyses of in-frame stop codons in protein-coding genes of Blastocrithidia nonstop with all three stop codons reassigned reveal a mechanism for UGA reassignment in eukaryotes involving shortening of the tRNA anticodon stem and a mutant eRF1 release factor.