title: RF1 knockout allows ribosomal incorporation of unnatural amino acids at multiple sites.
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.
authors.affiliation: The Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, La Jolla, California, USA.
authors.forename: David B F
authors.initials: DB
authors.lastname: Johnson
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Xu
authors.forename: Zhouxin
authors.initials: Z
authors.lastname: Shen
authors.forename: Jeffrey K
authors.initials: JK
authors.lastname: Takimoto
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Schultz
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Schmitz
authors.forename: Zheng
authors.initials: Z
authors.lastname: Xiang
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Ecker
authors.forename: Steven P
authors.initials: SP
authors.lastname: Briggs
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D018388:Codon, Terminator; D004926:Escherichia coli / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D023281:Genomics; D008958:Models, Molecular; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; C489005:prfA protein, E coli