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  • Review Article
  • Published:

Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology

Nature Reviews Microbiology volume 13, pages 707–721 (2015)Cite this article

Subjects

Key Points

  • The genetic code is not frozen.

  • Genetic code variations found in microorganisms include codon bias, codon reassignment, ambiguous decoding and natural genetic code expansion.

  • Codon bias, which is present in all sequenced genomes, modulates the rate of protein synthesis, which in turn regulates many biological processes.

  • Codon reassignment involves distinct mechanisms to completely redefine codon meaning.

  • Ambiguous decoding, which is detrimental at high levels, can be advantageous under stress conditions by increasing proteome diversity and activating stress responses.

  • Natural genetic code expansion with selenocysteine and pyrrolysine enables certain organisms to synthesize proteins with 21 or 22 amino acids, with consequences for enzymatic and metabolic efficiency that are just beginning to be understood.

  • The impact of genetic code evolution on microbial physiology is an emerging field, which is ripe for new discoveries.

Abstract

The genetic code, initially thought to be universal and immutable, is now known to contain many variations, including biased codon usage, codon reassignment, ambiguous decoding and recoding. As a result of recent advances in the areas of genome sequencing, biochemistry, bioinformatics and structural biology, our understanding of genetic code flexibility has advanced substantially in the past decade. In this Review, we highlight the prevalence, evolution and mechanistic basis of genetic code variations in microorganisms, and we discuss how this flexibility of the genetic code affects microbial physiology.

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Figure 1: Mechanisms of genetic code flexibility.
Figure 2: Biased codon usage.
Figure 3: Codon reassignment.
Figure 4: Ambiguous decoding.
Figure 5: Expanding the genetic code with Sec and Pyl.

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References

  1. Nirenberg, M. et al. RNA codewords and protein synthesis, VII. On the general nature of the RNA code. Proc. Natl Acad. Sci. USA 53, 1161–1168 (1965).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Söll, D. et al. Studies on polynucleotides, XLIX. Stimulation of the binding of aminoacyl-sRNA's to ribosomes by ribotrinucleotides and a survey of codon assignments for 20 amino acids. Proc. Natl Acad. Sci. USA 54, 1378–1385 (1965).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Li, M. & Tzagoloff, A. Assembly of the mitochondrial membrane system: sequences of yeast mitochondrial valine and an unusual threonine tRNA gene. Cell 18, 47–53 (1979).

    Article  CAS  PubMed  Google Scholar 

  4. Macino, G., Coruzzi, G., Nobrega, F. G., Li, M. & Tzagoloff, A. Use of the UGA terminator as a tryptophan codon in yeast mitochondria. Proc. Natl Acad. Sci. USA 76, 3784–3785 (1979). First discovery of codon reassignment in microorganisms.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ambrogelly, A., Palioura, S. & Söll, D. Natural expansion of the genetic code. Nat. Chem. Biol. 3, 29–35 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Plotkin, J. B. & Kudla, G. Synonymous but not the same: the causes and consequences of codon bias. Nat. Rev. Genet. 12, 32–42 (2011).

    Article  CAS  PubMed  Google Scholar 

  7. Quax, T. E., Claassens, N. J., Söll, D. & van der Oost, J. Codon bias as a means to fine-tune gene expression. Mol. Cell 59, 149–161 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pan, T. Adaptive translation as a mechanism of stress response and adaptation. Annu. Rev. Genet. 47, 121–137 (2013). Excellent review on benefits of ambiguous decoding under stress conditions.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Atkins, J. F. & Baranov, P. V. The distinction between recoding and codon reassignment. Genetics 185, 1535–1536 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Atkins, J. F., Gesteland, R. F., Reid, B. R. & Anderson, C. W. Normal tRNAs promote ribosomal frameshifting. Cell 18, 1119–1131 (1979). One of the first studies to demonstrate frameshifting and genetic code variability.

    Article  CAS  PubMed  Google Scholar 

  11. Grantham, R., Gautier, C., Gouy, M., Mercier, R. & Pave, A. Codon catalog usage and the genome hypothesis. Nucleic Acids Res. 84, r49–r62 (1980).

    Google Scholar 

  12. Shabalina, S. A., Spiridonov, N. A. & Kashina, A. Sounds of silence: synonymous nucleotides as a key to biological regulation and complexity. Nucleic Acids Res. 41, 2073–2094 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Bulmer, M. The selection-mutation-drift theory of synonymous codon usage. Genetics 129, 897–907 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Li, G. W., Oh, E. & Weissman, J. S. The anti-Shine-Dalgarno sequence drives translational pausing and codon choice in bacteria. Nature 484, 538–541 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Subramaniam, A. R., Pan, T. & Cluzel, P. Environmental perturbations lift the degeneracy of the genetic code to regulate protein levels in bacteria. Proc. Natl Acad. Sci. USA 110, 2419–2424 (2013).

    Article  CAS  PubMed  Google Scholar 

  16. Goodman, D. B., Church, G. M. & Kosuri, S. Causes and effects of N-terminal codon bias in bacterial genes. Science 342, 475–479 (2013).

    Article  CAS  PubMed  Google Scholar 

  17. Subramaniam, A. R. et al. A serine sensor for multicellularity in a bacterium. eLife 2, e01501 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Xu, Y. et al. Non-optimal codon usage is a mechanism to achieve circadian clock conditionality. Nature 495, 116–120 (2013). Suggested that non-optimal codons are used as a regulatory mechanism.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhou, M. et al. Non-optimal codon usage affects expression, structure and function of clock protein FRQ. Nature 495, 111–115 (2013). Suggested that non-optimal codons are used as a regulatory mechanism.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lang, B. F., Lavrov, D., Beck, N. & Steinberg, S. V. in Organelle Genetics (ed. Bullerwell, C. E.) 431–474 (Springer, 2012).

    Book  Google Scholar 

  21. Sengupta, S., Yang, X. & Higgs, P. G. The mechanisms of codon reassignments in mitochondrial genetic codes. J. Mol. Evol. 64, 662–688 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Alfonzo, J. D., Blanc, V., Estevez, A. M., Rubio, M. A. & Simpson, L. C to U editing of the anticodon of imported mitochondrial tRNATrp allows decoding of the UGA stop codon in Leishmania tarentolae. EMBO J. 18, 7056–7062 (1999). Demonstrated that RNA editing is responsible for UGA reassignment in Leishmania tarentolae mitochondria.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Muramatsu, T. et al. A novel lysine-substituted nucleoside in the first position of the anticodon of minor isoleucine tRNA from Escherichia coli. J. Biol. Chem. 263, 9261–9267 (1988).

    CAS  PubMed  Google Scholar 

  24. Mandal, D. et al. Agmatidine, a modified cytidine in the anticodon of archaeal tRNAIle, base pairs with adenosine but not with guanosine. Proc. Natl Acad. Sci. USA 107, 2872–2877 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ikeuchi, Y. et al. Agmatine-conjugated cytidine in a tRNA anticodon is essential for AUA decoding in archaea. Nat. Chem. Biol. 6, 277–282 (2010).

    Article  CAS  PubMed  Google Scholar 

  26. Tomita, K. et al. Codon reading patterns in Drosophila melanogaster mitochondria based on their tRNA sequences: a unique wobble rule in animal mitochondria. Nucleic Acids Res. 27, 4291–4297 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Cantara, W. A., Murphy, F. V., Demirci, H. & Agris, P. F. Expanded use of sense codons is regulated by modified cytidines in tRNA. Proc. Natl Acad. Sci. USA 110, 10964–10969 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Marniemi, J. & Parkki, M. G. Radiochemical assay of glutathione S-epoxide transferase and its enhancement by phenobarbital in rat liver in vivo. Biochem. Pharmacol. 24, 1569–1572 (1975).

    Article  CAS  PubMed  Google Scholar 

  29. Su, D. et al. An unusual tRNAThr derived from tRNAHis reassigns in yeast mitochondria the CUN codons to threonine. Nucleic Acids Res. 39, 4866–4874 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ling, J., Daoud, R., Lajoie, M. J., Söll, D. & Lang, B. F. Natural reassignment of CUU and CUA sense codons to alanine in Ashbya mitochondria. Nucleic Acids Res. 42, 499–508 (2014).

    Article  CAS  PubMed  Google Scholar 

  31. Ling, J. et al. Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment. Proc. Natl Acad. Sci. USA 109, 3281–3286 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ivanova, N. N. et al. Stop codon reassignments in the wild. Science 344, 909–913 (2014). Uncovered widespread stop codon reassignment events in microorganisms.

    Article  CAS  PubMed  Google Scholar 

  33. Mukai, T. et al. Codon reassignment in the Escherichia coli genetic code. Nucleic Acids Res. 38, 8188–8195 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Johnson, D. B. et al. Release factor one is nonessential in Escherichia coli. ACS Chem. Biol. 7, 1337–1344 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Heinemann, I. U. et al. Enhanced phosphoserine insertion during Escherichia coli protein synthesis via partial UAG codon reassignment and release factor 1 deletion. FEBS Lett. 586, 3716–3722 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lajoie, M. J. et al. Genomically recoded organisms expand biological functions. Science 342, 357–360 (2013). Created the first synthetic microorganism with complete removal of UAG stop codons.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Mukai, T. et al. Highly reproductive Escherichia coli cells with no specific assignment to the UAG codon. Sci. Rep. 5, 9699 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Mukai, T. et al. Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli. Nucleic Acids Res. 43, http://dx.doi.org/10.1093/nar/gkv787 (2015).

  39. Aerni, H. R., Shifman, M. A., Rogulina, S., O'Donoghue, P. & Rinehart, J. Revealing the amino acid composition of proteins within an expanded genetic code. Nucleic Acids Res. 43, e8 (2015).

    Article  PubMed  CAS  Google Scholar 

  40. Hammerling, M. J. et al. Bacteriophages use an expanded genetic code on evolutionary paths to higher fitness. Nat. Chem. Biol. 10, 178–180 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Schultz, D. W. & Yarus, M. Transfer RNA mutation and the malleability of the genetic code. J. Mol. Biol. 235, 1377–1380 (1994).

    Article  CAS  PubMed  Google Scholar 

  42. Ling, J., Reynolds, N. & Ibba, M. Aminoacyl-tRNA synthesis and translational quality control. Annu. Rev. Microbiol. 63, 61–78 (2009).

    Article  CAS  PubMed  Google Scholar 

  43. Rodnina, M. V. & Wintermeyer, W. Ribosome fidelity: tRNA discrimination, proofreading and induced fit. Trends Biochem. Sci. 26, 124–130 (2001).

    Article  CAS  PubMed  Google Scholar 

  44. Zaher, H. S. & Green, R. Fidelity at the molecular level: lessons from protein synthesis. Cell 136, 746–762 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Netzer, N. et al. Innate immune and chemically triggered oxidative stress modifies translational fidelity. Nature 462, 522–526 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Mascarenhas, A. P., An, S., Rosen, A. E., Martinis, S. A. & Musier-Forsyth, K. in Protein Engineering (eds RajBhandary, U. L. & Köhrer, C.) 153–200 (Springer, 2008).

    Google Scholar 

  47. Roy, H., Ling, J., Alfonzo, J. & Ibba, M. Loss of editing activity during the evolution of mitochondrial phenylalanyl-tRNA synthetase. J. Biol. Chem. 280, 38186–38192 (2005).

    Article  CAS  PubMed  Google Scholar 

  48. Li, L. et al. Naturally occurring aminoacyl-tRNA synthetases editing-domain mutations that cause mistranslation in Mycoplasma parasites. Proc. Natl Acad. Sci. USA 108, 9378–9383 (2011). Suggested that Mycoplasma spp. may use ambiguous decoding to defend against the host immune response.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Yadavalli, S. S. & Ibba, M. Selection of tRNA charging quality control mechanisms that increase mistranslation of the genetic code. Nucleic Acids Res. 41, 1104–1112 (2013).

    Article  CAS  PubMed  Google Scholar 

  50. Bezerra, A. R. et al. Reversion of a fungal genetic code alteration links proteome instability with genomic and phenotypic diversification. Proc. Natl Acad. Sci. USA 110, 11079–11084 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Miranda, I. et al. Candida albicans CUG mistranslation is a mechanism to create cell surface variation. mBio 4, e00285-13 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Javid, B. et al. Mycobacterial mistranslation is necessary and sufficient for rifampicin phenotypic resistance. Proc. Natl Acad. Sci. USA 111, 1132–1137 (2014). Demonstrated that ambiguous decoding increases resistance to an antibiotic in mycobacteria.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Fan, Y. et al. Protein mistranslation protects bacteria against oxidative stress. Nucleic Acids Res. 43, 1740–1748 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Wu, J., Fan, Y. & Ling, J. Mechanism of oxidant-induced mistranslation by threonyl-tRNA synthetase. Nucleic Acids Res. 42, 6523–6531 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Crick, F. H. The Croonian lecture, 1966. The genetic code. Proc. R. Soc. Lond. B 167, 331–347 (1967).

    Article  CAS  PubMed  Google Scholar 

  56. Turanov, A. A. et al. Genetic code supports targeted insertion of two amino acids by one codon. Science 323, 259–261 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Arner, E. S. Selenoproteins — what unique properties can arise with selenocysteine in place of cysteine? Exp. Cell Res. 316, 1296–1303 (2010).

    Article  CAS  PubMed  Google Scholar 

  58. Kim, H. Y. & Gladyshev, V. N. Different catalytic mechanisms in mammalian selenocysteine- and cysteine-containing methionine-R-sulfoxide reductases. PLoS. Biol. 3, e375 (2005).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  59. Snider, G. W., Ruggles, E., Khan, N. & Hondal, R. J. Selenocysteine confers resistance to inactivation by oxidation in thioredoxin reductase: comparison of selenium and sulfur enzymes. Biochemistry 52, 5472–5481 (2013).

    Article  CAS  PubMed  Google Scholar 

  60. Metanis, N. & Hilvert, D. Natural and synthetic selenoproteins. Curr. Opin. Chem. Biol. 22, 27–34 (2014).

    Article  CAS  PubMed  Google Scholar 

  61. Yoshizawa, S. & Böck, A. The many levels of control on bacterial selenoprotein synthesis. Biochim. Biophys. Acta 1790, 1404–1414 (2009). An excellent review of bacterial selenoproteins.

    Article  CAS  PubMed  Google Scholar 

  62. Bröcker, M. J., Ho, J. M., Church, G. M., Söll, D. & O'Donoghue, P. Recoding the genetic code with selenocysteine. Angew. Chem. Int. Ed. Engl. 53, 319–323 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Aldag, C. et al. Rewiring translation for elongation factor Tu-dependent selenocysteine incorporation. Angew. Chem. Int. Ed. Engl. 52, 1441–1445 (2013).

    Article  CAS  PubMed  Google Scholar 

  64. Su, D. et al. How an obscure archaeal gene inspired the discovery of selenocysteine biosynthesis in humans. IUBMB Life 61, 35–39 (2009).

    Article  CAS  PubMed  Google Scholar 

  65. Thyer, R., Robotham, S. A., Brodbelt, J. S. & Ellington, A. D. Evolving tRNASec for efficient canonical incorporation of selenocysteine. J. Am. Chem. Soc. 137, 46–49 (2015).

    Article  CAS  PubMed  Google Scholar 

  66. Miller, C. et al. A synthetic tRNA for EF-Tu mediated selenocysteine incorporation in vivo and in vitro. FEBS Lett. 589, 2194–2199 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Haruna, K., Alkazemi, M. H., Liu, Y., Söll, D. & Englert, M. Engineering the elongation factor Tu for efficient selenoprotein synthesis. Nucleic Acids Res. 42, 9976–9983 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Xu, J., Croitoru, V., Rutishauser, D., Cheng, Q. & Arner, E. S. Wobble decoding by the Escherichia coli selenocysteine insertion machinery. Nucleic Acids Res. 41, 9800–9811 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Zhang, Y., Romero, H., Salinas, G. & Gladyshev, V. N. Dynamic evolution of selenocysteine utilization in bacteria: a balance between selenoprotein loss and evolution of selenocysteine from redox active cysteine residues. Genome Biol. 7, R94 (2006). Although many new sequences are now available, this is still the definitive resource that documents the evolutionary replacement of Cys with Sec residues in bacterial proteins.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  70. Zhang, Y. & Gladyshev, V. N. Trends in selenium utilization in marine microbial world revealed through the analysis of the global ocean sampling (GOS) project. PLoS Genet. 4, e1000095 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  71. Stadtman, T. C. Selenocysteine. Annu. Rev. Biochem. 65, 83–100 (1996).

    Article  CAS  PubMed  Google Scholar 

  72. Jormakka, M., Byrne, B. & Iwata, S. Formate dehydrogenase — a versatile enzyme in changing environments. Curr. Opin. Struct. Biol. 13, 418–423 (2003).

    Article  CAS  PubMed  Google Scholar 

  73. Axley, M. J., Böck, A. & Stadtman, T. C. Catalytic properties of an Escherichia coli formate dehydrogenase mutant in which sulfur replaces selenium. Proc. Natl Acad. Sci. USA 88, 8450–8454 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Stock, T. & Rother, M. Selenoproteins in Archaea and Gram-positive bacteria. Biochim. Biophys. Acta 1790, 1520–1532 (2009).

    Article  CAS  PubMed  Google Scholar 

  75. Stadtman, T. C., Davis, J. N., Zehelein, E. & Böck, A. Biochemical and genetic analysis of Salmonella typhimurium and Escherichia coli mutants defective in specific incorporation of selenium into formate dehydrogenase and tRNAs. Biofactors 2, 35–44 (1989).

    CAS  PubMed  Google Scholar 

  76. Tetteh, A. Y. et al. Transcriptional response of selenopolypeptide genes and selenocysteine biosynthesis machinery genes in Escherichia coli during selenite reduction. Int. J. Microbiol. 2014, 394835 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  77. Lu, J. & Holmgren, A. The thioredoxin antioxidant system. Free Radic. Biol. Med. 66, 75–87 (2014).

    Article  CAS  PubMed  Google Scholar 

  78. Rother, M. & Krzycki, J. A. Selenocysteine, pyrrolysine, and the unique energy metabolism of methanogenic archaea. Archaea 2010, 453642 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  79. Stock, T., Selzer, M. & Rother, M. In vivo requirement of selenophosphate for selenoprotein synthesis in archaea. Mol. Microbiol. 75, 149–160 (2010).

    Article  CAS  PubMed  Google Scholar 

  80. Kryukov, G. V. & Gladyshev, V. N. The prokaryotic selenoproteome. EMBO Rep. 5, 538–543 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Cobucci-Ponzano, B., Rossi, M. & Moracci, M. Translational recoding in archaea. Extremophiles 16, 793–803 (2012).

    Article  CAS  PubMed  Google Scholar 

  82. Rother, M., Mathes, I., Lottspeich, F. & Böck, A. Inactivation of the selB gene in Methanococcus maripaludis: effect on synthesis of selenoproteins and their sulfur-containing homologs. J. Bacteriol. 185, 107–114 (2003). A seminal study on the phenotypic impact of removing Sec from the genetic code of the model archaeaon M. maripaludis.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Hohn, M. J., Palioura, S., Su, D., Yuan, J. & Söll, D. Genetic analysis of selenocysteine biosynthesis in the archaeon Methanococcus maripaludis. Mol. Microbiol. 81, 249–258 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Hao, B. et al. A new UAG-encoded residue in the structure of a methanogen methyltransferase. Science 296, 1462–1466 (2002). Discovery of the twenty-second genetically encoded amino acid, Pyl.

    Article  CAS  PubMed  Google Scholar 

  85. Srinivasan, G., James, C. M. & Krzycki, J. A. Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA. Science 296, 1459–1462 (2002).

    Article  CAS  PubMed  Google Scholar 

  86. Borrel, G. et al. Genome sequence of 'Candidatus Methanomethylophilus alvus' Mx1201, a methanogenic archaeon from the human gut belonging to a seventh order of methanogens. J. Bacteriol. 194, 6944–6945 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Borrel, G. et al. Comparative genomics highlights the unique biology of Methanomassiliicoccales, a Thermoplasmatales-related seventh order of methanogenic archaea that encodes pyrrolysine. BMC Genomics 15, 679 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  88. Gaston, M. A., Zhang, L., Green-Church, K. B. & Krzycki, J. A. The complete biosynthesis of the genetically encoded amino acid pyrrolysine from lysine. Nature 471, 647–650 (2011). Elucidated activities of the biosynthetic route to Pyl.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Krzycki, J. A. The path of lysine to pyrrolysine. Curr. Opin. Chem. Biol. 17, 619–625 (2013).

    Article  CAS  PubMed  Google Scholar 

  90. Blight, S. K. et al. Direct charging of tRNACUA with pyrrolysine in vitro and in vivo. Nature 431, 333–335 (2004). Elucidated the mechanism of Pyl decoding.

    Article  CAS  PubMed  Google Scholar 

  91. Polycarpo, C. et al. An aminoacyl-tRNA synthetase that specifically activates pyrrolysine. Proc. Natl Acad. Sci. USA 101, 12450–12454 (2004). Elucidated the mechanism of Pyl decoding.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Ticak, T., Kountz, D. J., Girosky, K. E., Krzycki, J. A. & Ferguson, D. J. Jr. A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase. Proc. Natl Acad. Sci. USA 111, e4668–e4676 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Heinemann, I. U. et al. The appearance of pyrrolysine in tRNAHis guanylyltransferase by neutral evolution. Proc. Natl Acad. Sci. USA 106, 21103–21108 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. O'Donoghue, P. et al. Reducing the genetic code induces massive rearrangement of the proteome. Proc. Natl Acad. Sci. USA 111, 17206–17211 (2014). Provided proteome-level view of the phenotypic impact of removing Pyl from the genetic code of M. acetivorans.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Quitterer, F., List, A., Eisenreich, W., Bacher, A. & Groll, M. Crystal structure of methylornithine synthase (PylB): insights into the pyrrolysine biosynthesis. Angew. Chem. Int. Ed. Engl. 51, 1339–1342 (2012).

    Article  CAS  PubMed  Google Scholar 

  96. Alkalaeva, E. et al. Translation termination in pyrrolysine-utilizing archaea. FEBS Lett. 583, 3455–3460 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Longstaff, D. G., Blight, S. K., Zhang, L., Green-Church, K. B. & Krzycki, J. A. In vivo contextual requirements for UAG translation as pyrrolysine. Mol. Microbiol. 63, 229–241 (2007).

    Article  CAS  PubMed  Google Scholar 

  98. Freistroffer, D. V., Kwiatkowski, M., Buckingham, R. H. & Ehrenberg, M. The accuracy of codon recognition by polypeptide release factors. Proc. Natl Acad. Sci. USA 97, 2046–2051 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Mansell, J. B., Guevremont, D., Poole, E. S. & Tate, W. P. A dynamic competition between release factor 2 and the tRNASec decoding UGA at the recoding site of Escherichia coli formate dehydrogenase H. EMBO J. 20, 7284–7293 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Mahapatra, A. et al. Characterization of a Methanosarcina acetivorans mutant unable to translate UAG as pyrrolysine. Mol. Microbiol. 59, 56–66 (2006).

    Article  CAS  PubMed  Google Scholar 

  101. Krzycki, J. A. Function of genetically encoded pyrrolysine in corrinoid-dependent methylamine methyltransferases. Curr. Opin. Chem. Biol. 8, 484–491 (2004).

    Article  CAS  PubMed  Google Scholar 

  102. Oelgeschlager, E. & Rother, M. In vivo role of three fused corrinoid/methyl transfer proteins in Methanosarcina acetivorans. Mol. Microbiol. 72, 1260–1272 (2009).

    Article  PubMed  CAS  Google Scholar 

  103. Polycarpo, C. R. et al. Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase. FEBS Lett. 580, 6695–6700 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Prat, L. et al. Carbon source-dependent expansion of the genetic code in bacteria. Proc. Natl Acad. Sci. USA 109, 21070–21075 (2012). Demonstrated natural Pyl decoding in bacteria and revealed first example of dynamic genetic code expansion.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Jiang, R. & Krzycki, J. A. PylSn and the homologous N-terminal domain of pyrrolysyl-tRNA synthetase bind the tRNA that is essential for the genetic encoding of pyrrolysine. J. Biol. Chem. 287, 32738–32746 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Katayama, H., Nozawa, K., Nureki, O., Nakahara, Y. & Hojo, H. Pyrrolysine analogs as substrates for bacterial pyrrolysyl-tRNA synthetase in vitro and in vivo. Biosci. Biotechnol. Biochem. 76, 205–208 (2012).

    Article  CAS  PubMed  Google Scholar 

  107. Nozawa, K. et al. Pyrrolysyl-tRNA synthetase-tRNAPyl structure reveals the molecular basis of orthogonality. Nature 457, 1163–1167 (2009).

    Article  CAS  PubMed  Google Scholar 

  108. O'Donoghue, P., Ling, J., Wang, Y. S. & Söll, D. Upgrading protein synthesis for synthetic biology. Nat. Chem. Biol. 9, 594–598 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Liu, C. C. & Schultz, P. G. Adding new chemistries to the genetic code. Annu. Rev. Biochem. 79, 413–444 (2010).

    Article  CAS  PubMed  Google Scholar 

  110. Chin, J. W. Reprogramming the genetic code. EMBO J. 30, 2312–2324 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Johnson, J. A., Lu, Y. Y., Van Deventer, J. A. & Tirrell, D. A. Residue-specific incorporation of non-canonical amino acids into proteins: recent developments and applications. Curr. Opin. Chem. Biol. 14, 774–780 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Chin, J. W. Expanding and reprogramming the genetic code of cells and animals. Annu. Rev. Biochem. 83, 379–408 (2014). An excellent review on engineering protein synthesis for genetic code expansion in diverse expression systems.

    Article  CAS  PubMed  Google Scholar 

  113. Rovner, A. J. et al. Recoded organisms engineered to depend on synthetic amino acids. Nature 518, 89–93 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Mandell, D. J. et al. Biocontainment of genetically modified organisms by synthetic protein design. Nature 518, 55–60 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Campbell, J. H. et al. UGA is an additional glycine codon in uncultured SR1 bacteria from the human microbiota. Proc. Natl Acad. Sci. USA 110, 5540–5545 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Wang, H. H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Isaacs, F. J. et al. Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science 333, 348–353 (2011). First example of a genome engineered with 62 codons by mutation of all TAGs to TAA.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. Wiedenheft, B., Sternberg, S. H. & Doudna, J. A. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338 (2012).

    Article  CAS  PubMed  Google Scholar 

  120. Gibson, D. G. et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52–56 (2010). First demonstration of genome transplantation with a synthetic genome.

    Article  CAS  PubMed  Google Scholar 

  121. Annaluru, N. et al. Total synthesis of a functional designer eukaryotic chromosome. Science 344, 55–58 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Budiman, M. E. et al. Eukaryotic initiation factor 4a3 is a selenium-regulated RNA-binding protein that selectively inhibits selenocysteine incorporation. Mol. Cell 35, 479–489 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  123. Hohn, M. J., Park, H. S., O'Donoghue, P., Schnitzbauer, M. & Söll, D. Emergence of the universal genetic code imprinted in an RNA record. Proc. Natl Acad. Sci. USA 103, 18095–18100 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Bilokapic, S. et al. Structure of the unusual seryl-tRNA synthetase reveals a distinct zinc-dependent mode of substrate recognition. EMBO J. 25, 2498–2509 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. Itoh, Y. et al. Decameric SelA•tRNASec ring structure reveals mechanism of bacterial selenocysteine formation. Science 340, 75–78 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  126. Sherrer, R. L., O'Donoghue, P. & Söll, D. Characterization and evolutionary history of an archaeal kinase involved in selenocysteinyl-tRNA formation. Nucleic Acids Res. 36, 1247–1259 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  127. Carlson, B. A. et al. Identification and characterization of phosphoseryl-tRNA[Ser]Sec kinase. Proc. Natl Acad. Sci. USA 101, 12848–12853 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  128. Palioura, S., Sherrer, R. L., Steitz, T. A., Söll, D. & Simonovic, M. The human SepSecS–tRNASec complex reveals the mechanism of selenocysteine formation. Science 325, 321–325 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Copeland, P. R., Fletcher, J. E., Carlson, B. A., Hatfield, D. L. & Driscoll, D. M. A novel RNA binding protein, SBP2, is required for the translation of mammalian selenoprotein mRNAs. EMBO J. 19, 306–314 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  130. Bifano, A. L., Atassi, T., Ferrara, T. & Driscoll, D. M. Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element. BMC Mol. Biol. 14, 12 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Allmang, C., Wurth, L. & Krol, A. The selenium to selenoprotein pathway in eukaryotes: more molecular partners than anticipated. Biochim. Biophys. Acta 1790, 1415–1423 (2009).

    Article  CAS  PubMed  Google Scholar 

  132. Yoshizawa, S. et al. Structural basis for mRNA recognition by elongation factor SelB. Nat. Struct. Mol. Biol. 12, 198–203 (2005).

    Article  CAS  PubMed  Google Scholar 

  133. Yuan, J. et al. Distinct genetic code expansion strategies for selenocysteine and pyrrolysine are reflected in different aminoacyl-tRNA formation systems. FEBS Lett. 584, 342–349 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. Eggertsson, G. & Söll, D. Transfer ribonucleic acid-mediated suppression of termination codons in Escherichia coli. Microbiol. Rev. 52, 354–374 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  135. Ambrogelly, A. et al. Pyrrolysine is not hardwired for cotranslational insertion at UAG codons. Proc. Natl Acad. Sci. USA 104, 3141–3146 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. Kavran, J. M. et al. Structure of pyrrolysyl-tRNA synthetase, an archaeal enzyme for genetic code innovation. Proc. Natl Acad. Sci. USA 104, 11268–11273 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  137. Wan, W. et al. A facile system for genetic incorporation of two different noncanonical amino acids into one protein in Escherichia coli. Angew. Chem. Int. Ed. Engl. 49, 3211–3214 (2010).

    Article  CAS  PubMed  Google Scholar 

  138. Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Genetically encoding Nɛ-acetyllysine in recombinant proteins. Nat. Chem. Biol. 4, 232–234 (2008).

    Article  CAS  PubMed  Google Scholar 

  139. Umehara, T. et al. N-acetyl lysyl-tRNA synthetases evolved by a CcdB-based selection possess N-acetyl lysine specificity in vitro and in vivo. FEBS Lett. 586, 729–733 (2012).

    Article  CAS  PubMed  Google Scholar 

  140. Yanagisawa, T., Umehara, T., Sakamoto, K. & Yokoyama, S. Expanded genetic code technologies for incorporating modified lysine at multiple sites. Chembiochem 15, 2181–2187 (2014).

    Article  CAS  PubMed  Google Scholar 

  141. Guo, L. T. et al. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution. Proc. Natl Acad. Sci. USA 111, 16724–16729 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  142. Lobanov, A. V. et al. Evolutionary dynamics of eukaryotic selenoproteomes: large selenoproteomes may associate with aquatic life and small with terrestrial life. Genome Biol. 8, r198 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  143. Grobe, T., Reuter, M., Gursinsky, T., Sohling, B. & Andreesen, J. R. Peroxidase activity of selenoprotein GrdB of glycine reductase and stabilisation of its integrity by components of proprotein GrdE from Eubacterium acidaminophilum. Arch. Microbiol. 187, 29–43 (2007).

    Article  PubMed  CAS  Google Scholar 

  144. Hurley, J. M. & Dunlap, J. C. Cell biology: a fable of too much too fast. Nature 495, 57–58 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

Work in the authors' laboratories was supported by grants from the US National Institute of General Medical Sciences (GM022854 to D.S.; and GM115431 to J.L.), from the Natural Sciences and Engineering Research Council of Canada (RGPIN 04282–2014 to P.O.), from the Canadian Institutes of Health Research Tier 2 Canada Research Chair (950-229917 to P.O.), and from The University of Texas Health Science Center at Houston start-up fund (to J.L.). The authors are grateful to I. Heinemann for discussions and a critical reading of the manuscript.

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Author notes

  1. Jiqiang Ling and Patrick O'Donoghue: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, 77030, Texas, USA

    Jiqiang Ling

  2. Department of Biochemistry, The University of Western Ontario, London, N6A 5C1, Ontario, Canada

    Patrick O'Donoghue

  3. Department of Chemistry, The University of Western Ontario, London, N6A 5C1, Ontario, Canada

    Patrick O'Donoghue

  4. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, 06520–8114, Connecticut, USA

    Dieter Söll

  5. Department of Chemistry, Yale University, New Haven, 06520–8114, Connecticut, USA

    Dieter Söll

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  1. Jiqiang Ling
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Supplementary information

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Genetic code flexibility in microorganisms (PDF 215 kb)

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Glossary

Aminoacyl-tRNA

(aa-tRNA). A tRNA molecule with an amino acid attached to the 3′ end. It is used as a substrate by the ribosome to synthesize proteins.

Codon–anticodon pairing

During translation, the bases of the mRNA codon and the tRNA anticodon need to match each other. Watson–Crick pairing (A–U and G–C) in the first and second positions of the codon is required for efficient decoding, whereas the third position allows more flexible pairing, for example, between G and U or using modified bases.

Synonymous codons

Different triplet nucleotide sequences that decode the same amino acid.

tRNA isoacceptors

Different tRNA species recognized by the same aminoacyl-tRNA synthetase and ligated with the same amino acid.

Misacylation

Incorrect pairing of an amino acid and tRNA by an aminoacyl-tRNA synthetase. Errors resulting from misacylation, if left uncorrected, reduce the overall translational fidelity.

Frameshifting

Change in the reading frame during translation due to mutations in the DNA, errors during transcription or translation or specific mRNA structures, leading to new protein sequences.

Shine–Dalgarno-like sequences

mRNA sequences that share high similarity with the Shine–Dalgarno sequence, which pairs with the anti-Shine–Dalgarno sequence of the ribosomal RNA.

Codon adaptation index

A method for analysing usage bias of synonymous codons using a set of highly expressed genes from a species as a reference to assign a score to each gene.

Wobble position

The third position of a codon, which is more flexibly recognized by the tRNA compared with other positions.

Phages

(Also called bacteriophages). Viruses that infect and propagate within bacteria. Phages contain their own genome but hijack the translational machinery of the bacterial host for protein synthesis.

RpoB

The β-subunit of the bacterial RNA polymerase and target of the antibiotic rifampicin.

Nucleophilicity

The property to donate an electron in chemical reactions.

Elongation factor Tu

(EF-Tu). A bacterial elongation factor that delivers aminoacyl-tRNAs to the ribosome during peptide synthesis. The counterpart of EF-Tu in archaea and eukaryotes is EF-1A.

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Ling, J., O'Donoghue, P. & Söll, D. Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology. Nat Rev Microbiol 13, 707–721 (2015). https://doi.org/10.1038/nrmicro3568

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