Skip to main content
SpringerLink
Log in

Artificial Division of Codon Boxes for Expansion of the Amino Acid Repertoire of Ribosomal Polypeptide Synthesis

  • Protocol
  • First Online:
Noncanonical Amino Acids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1728))

Abstract

In ribosomal polypeptide synthesis, the 61 sense codons redundantly code for the 20 proteinogenic amino acids. The genetic code contains eight family codon boxes consisting of synonymous codons that redundantly code for the same amino acid. Here, we describe the protocol of a recently published method to artificially divide such family codon boxes and encode multiple nonproteinogenic amino acids in addition to the 20 proteinogenic ones in a reprogrammed genetic code. To achieve this, an in vitro translation system reconstituted with 32 in vitro transcribed tRNASNN’s (S = C or G; N = U, C, A or G) was first developed, where the 32 tRNA transcripts can be charged with 20 proteinogenic amino acids by aminoacyl-tRNA synthetases in situ and orthogonally decode the corresponding 31 NNS sense codons as well as the AUG initiation codon. When some redundant tRNAGNN’s are replaced with tRNAGNN’s precharged with nonproteinogenic amino acids by means of flexizymes, the nonproteinogenic and proteinogenic aminoacyl-tRNAs can decode the NNC and NNG codons in the same family codon box independently. In this protocol, we describe expression of model peptides, including a macrocyclic peptide containing three kinds of N-methyl-amino acids reassigned to the vacant codons generated by the method of artificial division of codon boxes.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 79.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 99.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 139.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Murakami H, Ohta A, Ashigai H, Suga H (2006) A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nat Methods 3(5):357–359

    Article  CAS  PubMed  Google Scholar 

  2. Goto Y, Ohta A, Sako Y, Yamagishi Y, Murakami H, Suga H (2008) Reprogramming the translation initiation for the synthesis of physiologically stable cyclic peptides. ACS Chem Biol 3(2):120–129

    Article  CAS  PubMed  Google Scholar 

  3. Goto Y, Murakami H, Suga H (2008) Initiating translation with D-amino acids. RNA 14(7):1390–1398

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kawakami T, Murakami H, Suga H (2008) Messenger RNA-programmed incorporation of multiple N-methyl-amino acids into linear and cyclic peptides. Chem Biol 15(1):32–42

    Article  CAS  PubMed  Google Scholar 

  5. Xiao H, Murakami H, Suga H, Ferre-D'Amare AR (2008) Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme. Nature 454(7202):358–361

    Article  CAS  PubMed  Google Scholar 

  6. Ohta A, Murakami H, Higashimura E, Suga H (2007) Synthesis of polyester by means of genetic code reprogramming. Chem Biol 14(12):1315–1322

    Article  CAS  PubMed  Google Scholar 

  7. Goto Y, Katoh T, Suga H (2011) Flexizymes for genetic code reprogramming. Nat Protoc 6(6):779–790

    Article  CAS  PubMed  Google Scholar 

  8. Forster AC, Tan Z, Nalam MN, Lin H, Qu H, Cornish VW, Blacklow SC (2003) Programming peptidomimetic syntheses by translating genetic codes designed de novo. Proc Natl Acad Sci U S A 100(11):6353–6357

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Josephson K, Hartman MC, Szostak JW (2005) Ribosomal synthesis of unnatural peptides. J Am Chem Soc 127(33):11727–11735

    Article  CAS  PubMed  Google Scholar 

  10. Yamagishi Y, Shoji I, Miyagawa S, Kawakami T, Katoh T, Goto Y, Suga H (2011) Natural product-like macrocyclic N-methyl-peptide inhibitors against a ubiquitin ligase uncovered from a ribosome-expressed de novo library. Chem Biol 18(12):1562–1570

    Article  CAS  PubMed  Google Scholar 

  11. Nemoto N, Miyamoto-Sato E, Husimi Y, Yanagawa H (1997) In vitro virus: bonding of mRNA bearing puromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro. FEBS Lett 414(2):405–408

    Article  CAS  PubMed  Google Scholar 

  12. Roberts RW, Szostak JW (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc Natl Acad Sci U S A 94(23):12297–12302

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Terasaka N, Suga H (2014) Flexizymes-facilitated genetic code reprogramming leading to the discovery of drug-like peptides. Chem Lett 43(1):11–19

    Article  CAS  Google Scholar 

  14. Tanaka Y, Hipolito CJ, Maturana AD, Ito K, Kuroda T, Higuchi T, Katoh T, Kato HE, Hattori M, Kumazaki K, Tsukazaki T, Ishitani R, Suga H, Nureki O (2013) Structural basis for the drug extrusion mechanism by a MATE multidrug transporter. Nature 496(7444):247–251

    Article  CAS  PubMed  Google Scholar 

  15. Hayashi Y, Morimoto J, Suga H (2012) In vitro selection of anti-Akt2 thioether-macrocyclic peptides leading to isoform-selective inhibitors. ACS Chem Biol 7(3):607–613

    Article  CAS  PubMed  Google Scholar 

  16. Morimoto J, Hayashi Y, Suga H (2012) Discovery of macrocyclic peptides armed with a mechanism-based warhead: isoform-selective inhibition of human deacetylase SIRT2. Angew Chem Int Ed Engl 51(14):3423–3427

    Article  CAS  PubMed  Google Scholar 

  17. Yamagata K, Goto Y, Nishimasu H, Morimoto J, Ishitani R, Dohmae N, Takeda N, Nagai R, Komuro I, Suga H, Nureki O (2014) Structural basis for potent inhibition of SIRT2 deacetylase by a macrocyclic peptide inducing dynamic structural change. Structure 22(2):345–352

    Article  CAS  PubMed  Google Scholar 

  18. Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T (2001) Cell-free translation reconstituted with purified components. Nat Biotechnol 19(8):751–755

    Article  CAS  PubMed  Google Scholar 

  19. Komine Y, Adachi T, Inokuchi H, Ozeki H (1990) Genomic organization and physical mapping of the transfer RNA genes in Escherichia coli K12. J Mol Biol 212(4):579–598

    Article  CAS  PubMed  Google Scholar 

  20. FVt M, Ramakrishnan V (2004) Structure of a purine-purine wobble base pair in the decoding center of the ribosome. Nat Struct Mol Biol 11(12):1251–1252

    Article  Google Scholar 

  21. Gustilo EM, Vendeix FA, Agris PF (2008) tRNA's modifications bring order to gene expression. Curr Opin Microbiol 11(2):134–140. https://doi.org/10.1016/j.mib.2008.02.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Crick FH (1966) Codon—anticodon pairing: the wobble hypothesis. J Mol Biol 19(2):548–555

    Article  CAS  PubMed  Google Scholar 

  23. Iwane Y, Hitomi A, Murakami H, Katoh T, Goto Y, Suga H (2016) Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes. Nat Chem 8(4):317–325

    Article  CAS  PubMed  Google Scholar 

  24. Milligan JF, Groebe DR, Witherell GW, Uhlenbeck OC (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res 15(21):8783–8798

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Schagger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166(2):368–379

    Article  CAS  PubMed  Google Scholar 

  26. Schagger H (2006) Tricine-SDS-PAGE. Nat Protoc 1(1):16–22

    Article  PubMed  Google Scholar 

  27. Christian EL, McPheeters DS, Harris ME (1998) Identification of individual nucleotides in the bacterial ribonuclease P ribozyme adjacent to the pre-tRNA cleavage site by short-range photo-cross-linking. Biochemistry 37(50):17618–17628

    Article  CAS  PubMed  Google Scholar 

  28. Guo X, Campbell FE, Sun L, Christian EL, Anderson VE, Harris ME (2006) RNA-dependent folding and stabilization of C5 protein during assembly of the E. coli RNase P holoenzyme. J Mol Biol 360(1):190–203

    Article  CAS  PubMed  Google Scholar 

  29. Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75(3):663–670

    Article  CAS  PubMed  Google Scholar 

  30. Tamura K, Himeno H, Asahara H, Hasegawa T, Shimizu M (1992) In vitro study of E.coli tRNAArg and tRNALys identity elements. Nucleic Acids Res 20(9):2335–2339

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Sylvers LA, Rogers KC, Shimizu M, Ohtsuka E, Soll D (1993) A 2-thiouridine derivative in tRNAGlu is a positive determinant for aminoacylation by Escherichia coli glutamyl-tRNA synthetase. Biochemistry 32(15):3836–3841

    Article  CAS  PubMed  Google Scholar 

  32. Nureki O, Niimi T, Muramatsu T, Kanno H, Kohno T, Florentz C, Giege R, Yokoyama S (1994) Molecular recognition of the identity-determinant set of isoleucine transfer RNA from Escherichia coli. J Mol Biol 236(3):710–724

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgment

This research was supported by the Japan Science and Technology Agency (JST) Core Research for Evolutional Science and Technology (CREST) of Molecular Technologies to H.S., Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Young Scientists (B) to Y.G. (22750145), and Grants-in-Aid for JSPS Fellows to Y.I. (26-9576).

Author information

Authors and Affiliations

  1. Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan

    Yoshihiko Iwane, Takayuki Katoh, Yuki Goto & Hiroaki Suga

  2. JST-CREST, Tokyo, Japan

    Hiroaki Suga

Authors
  1. Yoshihiko Iwane
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Takayuki Katoh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuki Goto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hiroaki Suga
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hiroaki Suga .

Editor information

Editors and Affiliations

  1. Structural and Computational Biology Unit & Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany

    Edward A. Lemke

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Iwane, Y., Katoh, T., Goto, Y., Suga, H. (2018). Artificial Division of Codon Boxes for Expansion of the Amino Acid Repertoire of Ribosomal Polypeptide Synthesis. In: Lemke, E. (eds) Noncanonical Amino Acids. Methods in Molecular Biology, vol 1728. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7574-7_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7574-7_2

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7573-0

  • Online ISBN: 978-1-4939-7574-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation