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Nat Chem Biol. 2011 Sep 18;7(11):779-86. doi: 10.1038/nchembio.657.

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

Author information

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

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.

PMID:
21926996
PMCID:
PMC3201715
DOI:
10.1038/nchembio.657
[Indexed for MEDLINE]
Free PMC Article

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