title: In vitro biosynthesis of a universal t6A tRNA modification in Archaea and Eukarya.
abstract: N(6)-threonylcarbamoyladenosine (t(6)A) is a modified nucleotide found in all transfer RNAs (tRNAs) decoding codons starting with adenosine. Its role is to facilitate codon-anticodon pairing and to prevent frameshifting during protein synthesis. Genetic studies demonstrated that two universal proteins, Kae1/YgjD and Sua5/YrdC, are necessary for t(6)A synthesis in Saccharomyces cerevisiae and Escherichia coli. In Archaea and Eukarya, Kae1 is part of a conserved protein complex named kinase, endopeptidase and other proteins of small size (KEOPS), together with three proteins that have no bacterial homologues. Here, we reconstituted for the first time an in vitro system for t(6)A modification in Archaea and Eukarya, using purified KEOPS and Sua5. We demonstrated binding of tRNAs to archaeal KEOPS and detected two distinct adenosine triphosphate (ATP)-dependent steps occurring in the course of the synthesis. Our data, together with recent reconstitution of an in vitro bacterial system, indicated that t(6)A cannot be catalysed by Sua5/YrdC and Kae1/YgjD alone but requires accessory proteins that are not universal. Remarkably, we observed interdomain complementation when bacterial, archaeal and eukaryotic proteins were combined in vitro, suggesting a conserved catalytic mechanism for the biosynthesis of t(6)A in nature. These findings shed light on the reaction mechanism of t(6)A synthesis and evolution of molecular systems that promote translation fidelity in present-day cells.
authors.affiliation: Institut de Génétique et Microbiologie, Université Paris-Sud, IFR115, UMR8621-CNRS, 91405 Orsay, France.
authors.forename: Ludovic
authors.initials: L
authors.lastname: Perrochia
authors.forename: Estelle
authors.initials: E
authors.lastname: Crozat
authors.forename: Arnaud
authors.initials: A
authors.lastname: Hecker
authors.forename: Wenhua
authors.initials: W
authors.lastname: Zhang
authors.forename: Joseph
authors.initials: J
authors.lastname: Bareille
authors.forename: Bruno
authors.initials: B
authors.lastname: Collinet
authors.forename: Herman
authors.initials: H
authors.lastname: van Tilbeurgh
authors.forename: Patrick
authors.initials: P
authors.lastname: Forterre
authors.forename: Tamara
authors.initials: T
authors.lastname: Basta
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019843:Archaeal Proteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D055162:Biocatalysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D043903:Pyrococcus abyssi / Q000201:enzymology*; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C509947:KEOPS complex, S cerevisiae; D016601:RNA-Binding Proteins; C541142:SUA5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C003268:N(6)-(N-threonylcarbonyl)adenosine; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D011494:Protein Kinases; D010450:Endopeptidases; D000241:Adenosine