Article | Published:

RF1 knockout allows ribosomal incorporation of unnatural amino acids at multiple sites

Nature Chemical Biology volume 7, pages 779786 (2011) | Download Citation

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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.

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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.

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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

Author notes

    • David B F Johnson
    •  & Jianfeng Xu

    These authors contributed equally to this work.

Affiliations

  1. The Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, La Jolla, California, USA.

    • David B F Johnson
    • , Jianfeng Xu
    • , Jeffrey K Takimoto
    • , Zheng Xiang
    •  & Lei Wang
  2. Section of Cell and Development Biology, University of California, San Diego, La Jolla, California, USA.

    • Zhouxin Shen
    •  & Steven P Briggs
  3. Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA.

    • Matthew D Schultz
    • , Robert J Schmitz
    •  & Joseph R Ecker
  4. Graduate Bioinformatics Program, University of California, San Diego, La Jolla, California, USA.

    • Matthew D Schultz

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Lei Wang.

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DOI

https://doi.org/10.1038/nchembio.657

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