Stop codons have been exploited for genetic incorporation of unnatural amino acids (Uaas) in live cells, but the efficiency is low possibly due to competition from release factors, limiting the power and scope of this technology.  Here we show that the reportedly essential release factor 1 can be knocked out from Escherichia coli by fixing release factor 2.  The resultant strain JX33 is stable and independent, and reassigns UAG 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.  For instance, we selectively incorporated multiple p-acetyl-phenylalanine into glutathione S-transferase and multiple Ne-acetyl-lysine into histone H3a. 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 novel protein functions by enabling Uaa incorporation at multiple sites.

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

243rd National Meeting (2012)

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