1. 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
2. Department of Molecular Biophysics and Biochemistry,
3. Department of Chemistry, Yale University, New Haven, Connecticut 06520-8114, USA
4. Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
5. These authors contributed equally to this work.

Correspondence to: Dieter Söll^2,3Osamu Nureki^1,4 Correspondence and requests for materials should be addressed to O.N. (Email: nureki@ims.u-tokyo.ac.jp) or D.S. (Email: dieter.soll@yale.edu).

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 methylamines². Homologous methyltransferases and the Pyl biosynthetic and coding machinery are also found in two bacterial species^{1, 3}. 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 ancestor⁶; it then persisted in organisms that utilize methylamines as energy sources. Recent protein engineering efforts added non-canonical amino acids to the genetic code^{7, 8}. 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^{5, 9}. 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.

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