title: Pyrrolysyl-tRNA synthetase-tRNA(Pyl) structure reveals the molecular basis of orthogonality.
abstract: Pyrrolysine (Pyl), the 22nd natural amino acid, is genetically encoded by UAG and inserted into proteins by the unique suppressor tRNA(Pyl) (ref. 1). The Methanosarcinaceae produce Pyl and express Pyl-containing methyltransferases that allow growth on methylamines. Homologous methyltransferases and the Pyl biosynthetic and coding machinery are also found in two bacterial species. Pyl coding is maintained by pyrrolysyl-tRNA synthetase (PylRS), which catalyses the formation of Pyl-tRNA(Pyl) (refs 4, 5). Pyl is not a recent addition to the genetic code. PylRS was already present in the last universal common ancestor; it then persisted in organisms that utilize methylamines as energy sources. Recent protein engineering efforts added non-canonical amino acids to the genetic code. This technology relies on the directed evolution of an 'orthogonal' tRNA synthetase-tRNA pair in which an engineered aminoacyl-tRNA synthetase (aaRS) specifically and exclusively acylates the orthogonal tRNA with a non-canonical amino acid. For Pyl the natural evolutionary process developed such a system some 3 billion years ago. When transformed into Escherichia coli, Methanosarcina barkeri PylRS and tRNA(Pyl) function as an orthogonal pair in vivo. Here we show that Desulfitobacterium hafniense PylRS-tRNA(Pyl) is an orthogonal pair in vitro and in vivo, and present the crystal structure of this orthogonal pair. The ancient emergence of PylRS-tRNA(Pyl) allowed the evolution of unique structural features in both the protein and the tRNA. These structural elements manifest an intricate, specialized aaRS-tRNA interaction surface that is highly distinct from those observed in any other known aaRS-tRNA complex; it is this general property that underlies the molecular basis of orthogonality.
authors.affiliation: Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B34 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan.
authors.forename: Kayo
authors.initials: K
authors.lastname: Nozawa
authors.forename: Patrick
authors.initials: P
authors.lastname: O'Donoghue
authors.forename: Sarath
authors.initials: S
authors.lastname: Gundllapalli
authors.forename: Yuhei
authors.initials: Y
authors.lastname: Araiso
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Ishitani
authors.forename: Takuya
authors.initials: T
authors.lastname: Umehara
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
authors.forename: Osamu
authors.initials: O
authors.lastname: Nureki
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D049148:Aminoacylation; D018360:Crystallography, X-Ray; D045858:Desulfitobacterium / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D008239:Lysine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017021:Methanosarcina barkeri / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein
chemical_list: D012345:RNA, Transfer, Amino Acid-Specific; D000604:Amino Acyl-tRNA Synthetases; C456839:pyrrolysine; D008239:Lysine