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Title: Engineering posttranslational proofreading to discriminate nonstandard amino acids

Accurate incorporation of nonstandard amino acids (nsAAs) is central for genetic code expansion to increase the chemical diversity of proteins. However, aminoacyl-tRNA synthetases are polyspecific and facilitate incorporation of multiple nsAAs. We investigated and repurposed a natural protein degradation pathway, the N-end rule pathway, to devise an innovative system for rapid assessment of the accuracy of nsAA incorporation. Using this tool to monitor incorporation of the nsAA biphenylalanine allowed the identification of tyrosyl-tRNA synthetase (TyrRS) variants with improved amino acid specificity. The evolved TyrRS variants enhanced our ability to contain unwanted proliferation of genetically modified organisms. In conclusion, this posttranslational proofreading system will aid the evolution of orthogonal translation systems for specific incorporation of diverse nsAAs.
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  1. Harvard Medical School, Boston, MA (United States)
  2. Yale Univ., New Haven, CT (United States)
Publication Date:
Grant/Contract Number:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 115; Journal Issue: 3; Related Information: Supplementary information document on PNAS website.; Journal ID: ISSN 0027-8424
National Academy of Sciences, Washington, DC (United States)
Research Org:
Harvard Medical School, Boston, MA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23). Biological Systems Science Division; USDOE
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; genetic code expansion; protein degradation; N-end rule; nonstandard amino acids; synthetic biology
OSTI Identifier:
Alternate Identifier(s):
OSTI ID: 1416145