Abstract
Stop codons have been exploited for genetic incorporation of unnatural amino acids (Uaas) in live cells, but their low incorporation efficiency, which is possibly due to competition from release factors, limits the power and scope of this technology. Here we show that the reportedly essential release factor 1 (RF1) can be knocked out from Escherichia coli by 'fixing' release factor 2 (RF2). The resultant strain JX33 is stable and independent, and it allows UAG to be reassigned from a stop signal to an amino acid when a UAG-decoding tRNA-synthetase pair is introduced. Uaas were efficiently incorporated at multiple UAG sites in the same gene without translational termination in JX33. We also found that amino acid incorporation at endogenous UAG codons is dependent on RF1 and mRNA context, which explains why E. coli tolerates apparent global suppression of UAG. JX33 affords a unique autonomous host for synthesizing and evolving new protein functions by enabling Uaa incorporation at multiple sites.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Change history
27 September 2011
In the version of this article initially published online, the abbreviation for 'unnatural amino acid' (Uaa) was mistakenly used in text referring to the UAA stop codon. The errors have been corrected for the print, PDF and HTML versions of this article.
References
Osawa, S., Jukes, T., Watanabe, K. & Muto, A. Recent evidence for evolution of the genetic code. Microbiol. Rev. 56, 229–264 (1992).
Knight, R.D., Freeland, S.J. & Landweber, L.F. Rewiring the keyboard: evolvability of the genetic code. Nat. Rev. Genet. 2, 49–58 (2001).
Benzer, S. & Champe, S.P. A change from nonsense to sense in the genetic code. Proc. Natl. Acad. Sci. USA 48, 1114–1121 (1962).
Beier, H. & Grimm, M. Misreading of termination codons in eukaryotes by natural nonsense suppressor tRNAs. Nucleic Acids Res. 29, 4767–4782 (2001).
Wang, L., Brock, A., Herberich, B. & Schultz, P.G. Expanding the genetic code of Escherichia coli. Science 292, 498–500 (2001).
Wang, L. & Schultz, P.G. Expanding the genetic code. Angew. Chem. Int. Ed. Engl. 44, 34–66 (2004).
Wang, Q., Parrish, A.R. & Wang, L. Expanding the genetic code for biological studies. Chem. Biol. 16, 323–336 (2009).
Scolnick, E., Tompkins, R., Caskey, T. & Nirenberg, M. Release factors differing in specificity for terminator codons. Proc. Natl. Acad. Sci. USA 61, 768–774 (1968).
Rydén, S.M. & Isaksson, L. A temperature-sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and increased efficiency for some tRNA nonsense suppressors. Mol. Gen. Genet. 193, 38–45 (1984).
Gerdes, S.Y. et al. Experimental determination and system level analysis of essential genes in Escherichia coli MG1655. J. Bacteriol. 185, 5673–5684 (2003).
Nakamura, Y., Gojobori, T. & Ikemura, T. Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Res. 28, 292 (2000).
Moore, S.D. & Sauer, R.T. The tmRNA system for translational surveillance and ribosome rescue. Annu. Rev. Biochem. 76, 101–124 (2007).
Keiler, K.C., Waller, P.R. & Sauer, R.T. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science 271, 990–993 (1996).
Craigen, W.J., Cook, R.G., Tate, W.P. & Caskey, C.T. Bacterial peptide chain release factors: conserved primary structure and possible frameshift regulation of release factor 2. Proc. Natl. Acad. Sci. USA 82, 3616–3620 (1985).
Uno, M., Ito, K. & Nakamura, Y. Functional specificity of amino acid at position 246 in the tRNA mimicry domain of bacterial release factor 2. Biochimie 78, 935–943 (1996).
Pósfai, G. et al. Emergent properties of reduced-genome Escherichia coli. Science 312, 1044–1046 (2006).
Datsenko, K.A. & Wanner, B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640–6645 (2000).
Tischer, B.K., von Einem, J., Kaufer, B. & Osterrieder, N. Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. Biotechniques 40, 191–197 (2006).
Ito, K., Uno, M. & Nakamura, Y. Single amino acid substitution in prokaryote polypeptide release factor 2 permits it to terminate translation at all three stop codons. Proc. Natl. Acad. Sci. USA 95, 8165–8169 (1998).
Zhang, S., Ryden-Aulin, M., Kirsebom, L.A. & Isaksson, L.A. Genetic implication for an interaction between release factor one and ribosomal protein L7-L12 in vivo. J. Mol. Biol. 242, 614–618 (1994).
Dahlgren, A. & Ryden-Aulin, M. A novel mutation in ribosomal protein S4 that affects the function of a mutated RF1. Biochimie 82, 683–691 (2000).
Kaczanowska, M. & Ryden-Aulin, M. Temperature sensitivity caused by mutant release factor 1 is suppressed by mutations that affect 16S rRNA maturation. J. Bacteriol. 186, 3046–3055 (2004).
Wang, L., Zhang, Z., Brock, A. & Schultz, P.G. Addition of the keto functional group to the genetic code of Escherichia coli. Proc. Natl. Acad. Sci. USA 100, 56–61 (2003).
Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).
Wang, L., Xie, J., Deniz, A.A. & Schultz, P.G. Unnatural amino acid mutagenesis of green fluorescent protein. J. Org. Chem. 68, 174–176 (2003).
Huang, Y. et al. A convenient method for genetic incorporation of multiple noncanonical amino acids into one protein in Escherichia coli. Mol. Biosyst. 6, 683–686 (2010).
Chen, S., Schultz, P.G. & Brock, A. An improved system for the generation and analysis of mutant proteins containing unnatural amino acids in Saccharomyces cerevisiae. J. Mol. Biol. 371, 112–122 (2007).
Abe, H., Abo, T. & Aiba, H. Regulation of intrinsic terminator by translation in Escherichia coli: transcription termination at a distance downstream. Genes Cells 4, 87–97 (1999).
Ueda, K. et al. Bacterial SsrA system plays a role in coping with unwanted translational readthrough caused by suppressor tRNAs. Genes Cells 7, 509–519 (2002).
Takimoto, J.K., Adams, K.L., Xiang, Z. & Wang, L. Improving orthogonal tRNA-synthetase recognition for efficient unnatural amino acid incorporation and application in mammalian cells. Mol. Biosyst. 5, 931–934 (2009).
Schrader, J.M., Chapman, S.J. & Uhlenbeck, O.C. Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding. Proc. Natl. Acad. Sci. USA 108, 5215–5220 (2011).
Ledoux, S. & Uhlenbeck, O.C. Different aa-tRNAs are selected uniformly on the ribosome. Mol. Cell 31, 114–123 (2008).
Yokoyama, S. & Nishimura, S. Modified nucleosides and codon recognition in tRNA: Structure, Biosynthesis, and Function (eds. Soll, D. & RajBhandary, U.L.) 207–223 (ASM Press, 1995).
Kane, J.F. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr. Opin. Biotechnol. 6, 494–500 (1995).
Mukai, T. et al. Codon reassignment in the Escherichia coli genetic code. Nucleic Acids Res. 38, 8188–8195 (2010).
Richardson, S.M., Wheelan, S.J., Yarrington, R.M. & Boeke, J.D. GeneDesign: rapid, automated design of multikilobase synthetic genes. Genome Res. 16, 550–556 (2006).
Agafonov, D.E., Kolb, V.A. & Spirin, A.S. Ribosome-associated protein that inhibits translation at the aminoacyl-tRNA binding stage. EMBO Rep. 2, 399–402 (2001).
Lee, J.H., Yeo, W.S. & Roe, J.H. Induction of the sufA operon encoding Fe-S assembly proteins by superoxide generators and hydrogen peroxide: involvement of OxyR, IHF and an unidentified oxidant-responsive factor. Mol. Microbiol. 51, 1745–1755 (2004).
Luger, K., Rechsteiner, T.J. & Richmond, T.J. Preparation of nucleosome core particle from recombinant histones. Methods Enzymol. 304, 3–19 (1999).
Ossowski, S. et al. Sequencing of natural strains of Arabidopsis thaliana with short reads. Genome Res. 18, 2024–2033 (2008).
Acknowledgements
We are very grateful to R. Sauer (MIT) for providing strain X90 ssrA1::cat, to L. Isaksson (Stockholm University) for providing strain MRA8, to W. Liu (Texas A&M University) for providing plasmid pET-L11C and to J. Kadonaga (University of California San Diego) for suggestions on H3a purification. We thank V.K. Lacey for proofreading the manuscript. J.X. was partially funded by the Pioneer Fellowship. M.D.S. was supported by National Science Foundation IGERT training grant (DGE-0504645). R.J.S. was supported by an US National Institutes of Health National Research Service Award postdoctoral fellowship (F32-HG004830). J.R.E. acknowledges support from the Mary K. Chapman Foundation. L.W. acknowledges support from the Ray Thomas Edwards Foundation, Searle Scholar Program (06-l-119), Beckman Young Investigator Program, March of Dimes Foundation (#5-FY08-110), California Institute for Regenerative Medicine (RN1-00577-1) and US National Institutes of Health (1DP2OD004744-01).
Author information
Authors and Affiliations
Contributions
D.B.F.J. incorporated tyrosine and various Uaas into EGFP and histone H3a using JX33, characterized JX2.0 and JX3.0 strains with growth, western, fluorescence, and temperature-sensitive complementation assays, performed endogenous UAG suppression studies, analyzed the data and wrote the manuscript; J.X. generated the RF1-knockout strains and analyzed the data; Z.S. and S.P.B. characterized amino acid and Uaa incorporation with mass spectrometry, analyzed the data and wrote the mass spectrometry section; J.K.T. incorporated Uaa into GST, compared Uaa incorporation efficiency in JX33 and in BL21 expressing L11C and analyzed the data; M.D.S., R.J.S. and J.R.E. sequenced all E. coli strains described, analyzed the data and wrote the genomic sequencing section; Z.X. synthesized Uaas; L.W. conceived and directed the project, analyzed the data and wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Text and Figures
Supplementary Methods and Supplementary Results (PDF 2466 kb)
Rights and permissions
About this article
Cite this article
Johnson, D., Xu, J., Shen, Z. et al. RF1 knockout allows ribosomal incorporation of unnatural amino acids at multiple sites. Nat Chem Biol 7, 779–786 (2011). https://doi.org/10.1038/nchembio.657
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nchembio.657
This article is cited by
-
Reprogramming the genetic code
Nature Reviews Genetics (2021)
-
Multiplex suppression of four quadruplet codons via tRNA directed evolution
Nature Communications (2021)
-
Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency
Amino Acids (2021)
-
Expanding the enzyme universe with genetically encoded unnatural amino acids
Nature Catalysis (2020)
-
Non-canonical amino acid labeling in proteomics and biotechnology
Journal of Biological Engineering (2019)