Transfer RNA are central elements of the translational system and carry a large diversity of modified nucleosides (derived from canonical nucleosides A, U, G, and C), which tune the stability, the decoding capacity and the identity of these oligonucleotides. t6A (threonylcarbamoyl-N6- adenosine) is a hypermodified nucleoside found at the position 37 (next to the anticodon) in all tRNA decoding ANN codons. It plays an essential role in the fidelity of translation through two main functions: (i) it ensures a correct conformation of the anticodon loop; (ii) it enhances codon/anticodon pairing to prevent frameshifting during translation. This nucleoside is universal, found in Archaea, Bacteria, Eukarya and also in organites such as mitochondria, which suggests that it appeared early in the evolution, probably before the last universal common ancestor (LUCA). Despite the importance of t6A and its distribution, its biosynthetic pathway has remained unknown for almost 40 years.Recently, genetic studies have shown that two universal proteins, Sua5/YrdC and Kae1/YgjD, are both necessary for synthesis of t6A in Saccharomyces cerevisiae and Escherichia coli. In Bacteria, the in vitro synthesis of t6A requires two other bacterial specific proteins called YeaZ and YjeE. In Archaea and Eukarya, Kae1 (the YgjD orthologue) is a part of a conserved protein complex called KEOPS (for Kinase Endopeptidase and Other Proteins of Small size), with three other proteins Bud32, Cgi121 and Pcc1, that have no bacterial homologues. Since its discovery in 2006 in yeast, this complex has been involved in several cellular processes (telomere homeostasis, genome maintenance, transcription regulation), but its real function remained unclear.Using an in vitro biochemical approach we aimed to characterize and compare the t6A biosynthesis systems from the three domains of life, using as model organisms Pyrococcus abyssi (Archaea) Saccharomyces cerevisiae (Eukarya), and Escherichia coli (Bacteria). We have reconstituted for the first time an in vitro system for t6A modification in Archaea and Eukarya, using purified KEOPS and Sua5. This allowed us to propose a model for the catalytic mechanism, and using in vitro complementation experiments we demonstrated that this mechanism is universal: Sua5/YrdC orthologues are interchangeable, and the KEOPS complex is the functional analogue of the bacterial trio YeaZ/YgjD/YjeE. In the second part of this work we have studied the role of each sub unit in the synthesis of t6A. Using KEOPS from P. abyssi as model we demonstrated that Kae1 is the only catalytic component while the three other partners have distinct functions in dimerization, tRNA binding and allosteric regulation. Finally, we have focused on the t6A synthesis in the mitochondria of S.cerevisiae, and shown that Sua5 and Qri7, the mitochondrial orthologue of Kae1/YgjD, catalyze together the synthesis of t6A and so represent a minimal two-component system.Overall these findings shed light on the reaction mechanism of t6A synthesis in the three domains of life, and allowed proposing a scenario concerning the history of the t6A synthesis machinery and its evolution.
