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Dispatch R563 Protein synthesis: Twenty three amino acids and counting Michael Ibba*, Constantinos Stathopoulos† and Dieter Söll†‡ The genetic code can be interpreted during translation as 21 amino acids and three termination signals. Recent advances at the interface of chemistry and molecular biology are extending the genetic code to allow assignment of new amino acids to existing codons, providing new functional groups for protein synthesis. Addresses: *Center for Biomolecular Recognition, Department of Medical Biochemistry and Genetics, Laboratory B, The Panum Institute, Blegdamsvej 3c, DK-2200, Copenhagen N, Denmark. †Departments of Molecular Biophysics and Biochemistry, and ‡Chemistry, Yale University, New Haven, Connecticut 06520-8114, USA. E-mail: [email protected] Current Biology 2001, 11:R563–R565 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. From the days when the genetic code was first broken to its near-universal validation in the genomic age, the riddle has persisted as to why only twenty amino acids were selected for ribosomal protein synthesis and what governed the selection of this particular set. These twenty amino acids are phenomenally versatile building blocks, providing proteins which permit life to flourish in a wide variety of physically and chemically demanding habitats, from below 0°C to over 100°C. Even though over 120 additional amino acids are found in natural proteins [1], they are all outside the genetic code, being instead formed by post-translational modification of the 20 canonical amino acids of ribosomally made proteins. Thus, protein synthesis relies universally on 61 sense codons that encode 21 amino acids — including the nonstandard amino acid selenocysteine [2] — and three termination codons. Now, just as the integration of chemistry and molecular biology first facilitated the breaking of the genetic code in the 1960s, the application of these same disciplines four decades later has finally enabled the extension of the genetic code to include new amino acids [3,4]. Chemists and molecular biologists have long sought to alter the rules governing protein synthesis with the aim of inserting novel amino acids into proteins. Why other amino acids present in the cell, such as ornithine, have been excluded from the standard repertoire of coded protein synthesis remains a mystery. Two classes of biomolecules are entrusted with ensuring the fidelity of translation: transfer RNAs (tRNAs), which carry the messenger (m)RNA-encoded amino acids to the ribosome, and aminoacyl-tRNA synthetases, which acylate — ‘charge’ — the tRNA with the correct (cognate) amino acid. The specificity of this process is extremely high — the error frequency is in the range 10–4–10–5 [5] — as each mischarging error will lead to the formation of a mutant polypeptide. The fidelity of the acylation process is ensured by sophisticated editing mechanisms associated with many aminoacyl-tRNA synthetases, in which incorrectly activated or charged amino acids are removed (reviewed in [6]). Although these editing activities are a major barrier to the incorporation of nonstandard amino acids into proteins, it has been known for many years that chinks can be found in the cell’s armor. For instance, the leucine analog 5′,5′,5′-trifluoroleucine was shown to be charged onto tRNA and incorporated into proteins by Escherichia coli [7], while Bacillus subtilis growth could be made dependent on the tryptophan analog 4-fluorotryptophan to the virtual exclusion of the natural amino acid [8]. Furthermore, the in vivo production of proteins containing selenomethionine is a key tool of modern X-ray crystallography [9]. It was recognized early on that protein synthesis is at the interface of chemistry and biology, and attempts to deviate from the constraints of the genetic code in vitro began with the combined chemical/enzymatic generation of tRNAs acylated with desired amino acid derivatives [10]. The idea that nonsense codons are ideal signals for amino acid misincorporation mediated by suppressor tRNAs is well established [11]. When these suppressor tRNAs are chemically or enzymatically aminoacylated they can be used for the incorporation of synthetic amino acids at specific positions in proteins, but only in vitro or ex vivo [12,13]. The long search for an in vivo approach to site-specific cotranslational insertion of an unusual amino acid — effectively an expansion of the cellular genetic code — has come to a fruition in the work of Wang et al. [3]. They engineered E. coli for in vivo incorporation of O-methyl-Ltyrosine in response to an amber (UAG) nonsense codon positioned in-frame in the gene encoding dihydrofolate reductase. To this end it was necessary to introduce an orthogonal tRNA–aminoacyl-tRNA synthetase pair into E. coli, involving an engineered aminoacyl-tRNA synthetase that recognizes only the cognate orthogonal tRNA and a non-standard amino acid. To achieve the desired properties, the tRNA–aminoacyltRNA synthetase pair was imported from another organism, Methanococcus jannaschii, whose tyrosyl-tRNA synthetase does not charge E. coli tRNAs. The tRNA used was a suppressor tRNATyr which is not charged by E. coli aminoacyltRNA synthetases. In vivo selection was used to obtain an RNA–protein pair that interact as little as possible with R564 Current Biology Vol 11 No 14 Figure 1 M. jannaschii tRNATyr CUA E. coli tRNATyr Random mutagenesis No aminoacylation M. jannaschii mutRNATyr (Library) CUA Wild-type M. jannaschii TyrRS Negative selection Mutagenesis of active site residues M. jannaschii mutRNATyr CUA that cannot be used by E. coli AARSs Positive selection PCR-random mutagenesis Positive selection Pool of mutant M. jannaschii TyrRSs Orthogonal M. jannaschii mut tRNATyr CUA Aminoacylation Addition of non-natural AA Positive selection DNA shuffling Further selection Orthogonal tRNA–synthetase pair inside the cells Current Biology Schematic representation of the selection of an orthogonal tRNA–aminoacyl-tRNA pair for the in vivo site-specific incorporation of O-methyl-L-tyrosine in E. coli (see text for details). Separate selections were first applied to obtain a pool of mutant tyrosyl-tRNA synthetase variants and an orthogonal derivative of tRNATyrCUA (a variant of tRNATyr where the anticodon has been changed to generate an amber suppressor tRNA). These two components were then brought together and used to select particular tyrosyl-tRNA synthetase mutants that preferentially recognize O-methyl-L-tyrosine rather than tyrosine. TyrRS, tyrosyl-tRNA synthetase; AARS, aminoacyl-tRNA synthetase; AA, amino acid. E. coli tRNAs or tyrosine, but show the best interaction within the orthogonal pair and with the tyrosine analog (Figure 1). The goal was successfully achieved: the analog incorporation was over 99% faithful [3]. The successful generation of an orthogonal tRNA–aminoacyl-tRNA synthetase pair encompassed the use of a heterologous tRNA and synthetase and a suppressor tRNA (principles that have also been employed successfully and extended to eukaryotic systems by RajBhandary and colleagues [14,15]). Perhaps the key factor here, however, was the initial abolition of the original activities of the tRNA–synthetase by mutation, which then gave the subsequent selection of orthogonal activities and specificities a real chance to succeed. In effect, Wang et al. [3] ensured that they minimized the sequence space that had to be sampled at each step of their selection procedure. Another key point in this approach was the use of tyrosyltRNA synthetase, which does not have any known editing activity. The principles of this approach are not specific to a particular amino acid, and so can be extended to other synthetase–tRNA pairs on a case-by-case basis for the selection (from combinatorial libraries) of many structurally diverse amino-acid analogs. It is an interesting coincidence that tyrosyl-tRNA synthetase, the crucial synthetase used in this study, was the first enzyme to be subjected to structure-directed protein engineering twenty years ago [16]. In another recent study [4], an elegant genetic system was used to sample all E. coli codons in vivo to determine which may be used for ‘encoding’, not only their canonical amino acid, but also cysteine. This was based on a sensitive selection for ‘suppression’ of a mutation of the codon for an essential cysteine in the active site of thymidylate synthase. Mutations that caused such a phenotype were shown to map to the valS gene, which encodes valyltRNA synthetase. The mutations were found to affect residues in the editing site of the enzyme, so that the mutant enzymes were less efficient at correcting the misacylated Cys–tRNAVal. Aminobutyric acid, long known to be recognized by valyl-tRNA synthetase, was then fed to the growth medium in the hope it would be incorporated instead of valine. This was the case, 24% of the valines in the E. coli proteins being replaced by aminobutyric acid. While the observed incorporation of a significant amount of aminobutyric acid into protein was not surprising in light of the earlier knowledge, it was most revealing that the reason for the genetically selected facile incorporation was found to be in the reduced editing activity of an aminoacyl-tRNA synthetase. In effect, the approach taken by Döring et al. [4] makes a virtue of editing activities, seen as one of the limitations in the selection of candidates for orthogonal pair generation by Wang et al. [3], as a means of selecting for novel enzymatic activities. The immediate impact of these recent breakthroughs is that they provide new tools for protein engineering. The potential of such tools has long been appreciated from in vitro studies and can now be fully exploited in vivo. The broader finding that the amino acids of the genetic code are mutable, as predicted by Wong [17], has wider implications for our understandings of the evolution of this aspect of protein synthesis. As Döring et al. [4] point out, the infiltration of the genetic code by an additional amino acid now shows how a less stringent priomordial code could Dispatch have been restricted during evolution — an ancestral valyl-tRNA synthetase could in effect settle on a particular substrate from several choices. On the other hand, Wang et al. [3] reveal just how amenable the genetic code is to accommodating new amino acids, indicating how the genetic code may have evolved from encoding a handful of amino acids to the twenty routinely used today. A striking example of the ability of the protein synthesis machinery to find different ways to accommodate amino acids can still be seen in the case of cysteine. To date, four unrelated routes have been identified for the synthesis of Cys-tRNA: the conventional cysteinyl-tRNA synthetase [18], a dual-specificity prolyl-cysteinyl-tRNA synthetase [19], an unclassified evolutionarily restricted cysteinyltRNA synthetase [20] and a non-editing valyl-tRNA synthetase mutant [4]. In all but the last case, cysteine is specifically attached to cysteine-specific tRNA (tRNACys) — the variety of synthesis mechanisms does not impart any variety to the genetic code. While this raises the obvious question as to why so many routes service the genetic code with cysteine, to which there is currently no obvious answer, the question also arises as to why the catalytic promiscuity observed for cysteine has not been exploited to expand the genetic code to include more amino acids. A heretical answer would be that it has, and we simply have not yet found the ingenuity with which to look for new meanings in the genetic code. Another increasingly observed evolutionary trend, lateral gene transfer, would seem to provide a more credible answer as to why the genetic code is almost universal and confined to twenty amino acids. Rampant lateral gene transfer has been proposed as a key event in the evolution of the first organisms, with translation being one of the first cellular processes to become ‘fixed’ [21]. Once translation was established, its universal use of twenty amino acids would allow organisms to take full advantage of gene transfer to sample a large gene pool; any organism not conforming to this universal code would thus be at a disadvantage. Such an explanation extols the potential advantages of a universal genetic code, without in any way requiring twenty to be a magic number, a fact now finally disproved. Acknowledgements The authors would like to thank Uttam RajBhandary, William Whitman, and Carl Woese for stimulating discussions. MI is the recipient of an Investigator Fellowship from the Alfred Benzon Foundation. Work in the authors’ laboratories was supported by grants from the Novo Nordisk Foundation (MI), the Danish Research Agency (MI), the National Institute of General Medical Sciences (DS), National Aeronautics and Space Administration (DS), and the Department of Energy (DS). References 1. Uy R, Wold F: Posttranslational covalent modification of proteins. 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Wong JT: Membership mutation of the genetic code: loss of fitness by tryptophan. Proc Natl Acad Sci USA 1983, 80:6303-6306. 9. Hendrickson WA, Horton JR, LeMaster DM: Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure. EMBO J 1990, 9:1665-1672. 10. Hecht SM, Alford BL, Kuroda Y, Kitano S: ‘Chemical aminoacylation’ of tRNA’s. J Biol Chem 1978, 253:4517-4520. 11. Inokuchi H, Hoben P, Yamao F, Ozeki H, Söll D. Transfer RNA mischarging mediated by a mutant Escherichia coli glutaminyl-tRNA synthetase. Proc Natl Acad Sci USA 1984, 81:5076-5080. 12. Noren CJ, Anthony-Cahill SJ, Griffith MC, Schultz PG: A general method for site-specific incorporation of unnatural amino acids into proteins. Science 1989, 244:182-188. 13. Nowak MW, Kearney PC, Sampson JR, Saks ME, Labarca CG, Silverman SK, Zhong W, Thorson J, Abelson JN, Davidson N, et al.: Nicotinic receptor binding site probed with unnatural amino acid incorporation in intact cells. Science 1995, 268:439-442. 14. Drabkin HJ, Park HJ, RajBhandary UL: Amber suppression in mammalian cells dependent upon expression of an Escherichia coli aminoacyl-tRNA synthetase gene. Mol Cell Biol 1996, 16:907-913. 15. Kowal AK, Köhrer C, RajBhandary UL: Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria. Proc Natl Acad Sci USA 2001, 98:2268-2273. 16. Winter G, Fersht AR, Wilkinson AJ, Zoller M, Smith M: Redesigning enzyme structure by site-directed mutagenesis: tyrosyl tRNA synthetase and ATP binding. Nature 1982, 299:756-758. 17. Wong JT: Evolution of the genetic code. Microbiol Sci 1988, 5:174-181. 18. Li T, Graham DE, Stathopoulos C, Haney PJ, Kim HS, Vothknecht U, Kitabatake M, Hong KW, Eggertsson G, Curnow AW, Lin W, et al.: Cysteinyl-tRNA formation: the last puzzle of aminoacyl-tRNA synthesis. FEBS Lett 1999, 462:302-306. 19. Stathopoulos C, Li T, Longman R, Vothknecht UC, Becker HD, Ibba M, Söll D: One polypeptide with two aminoacyl-tRNA synthetase activities. Science 2000, 287:479-482. 20. Fabrega C, Farrow MA, Mukhopadhyay B, de Crécy-Lagard V, Ortiz AR, Schimmel P: An aminoacyl tRNA synthetase whose sequence fits into neither of the two known classes. Nature 2001, 411:110-114. 21. Woese C: The universal ancestor. Proc Natl Acad Sci USA 1998, 95:6854-6859.