Letters pubs.acs.org/acschemicalbiology Release Factor One Is Nonessential in Escherichia coli David B. F. Johnson,†,⊥ Chong Wang,†,⊥ Jianfeng Xu,† Joseph R. Ecker,‡,∥ and Lei Wang*,† Matthew D. Schultz, ‡,§ Robert J. Schmitz,‡ †The Jack H. Skirball Center for Chemical Biology and Proteomics and ‡Genomic Analysis Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, California 92037, United States §Bioinformatics Program, University of California at San Diego, La Jolla, California 92093, United States ∥Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, Maryland 20815, United States *S Supporting Information ABSTRACT: Recoding a stop codon to an amino acid may aﬀord orthogonal genetic systems for biosynthesizing new protein and organism properties. Although reassignment of stop codons has been found in extant organisms, a model organism is lacking to investigate the reassignment process and to direct code evolution. Complete reassignment of a stop codon is precluded by release factors (RFs), which recognize stop codons to terminate translation. Here we discovered that RF1 could be unconditionally knocked out from various Escherichia coli stains, demonstrating that the reportedly essential RF1 is generally dispensable for the E. coli species. The apparent essentiality of RF1 was found to be caused by the ineﬃciency of a mutant RF2 in terminating all UAA stop codons; a wild type RF2 was suﬃcient for RF1 knockout. The RF1- knockout strains were autonomous and unambiguously reassigned UAG to encode natural or unnatural amino acids (Uaas) at multiple sites, aﬀording a previously unavailable model for studying code evolution and a unique host for exploiting Uaas to evolve new biological functions. A all organisms, small deviations have been discovered, lthough the canonical genetic code is preserved in almost including the reassignment of sense codons from one amino acid to another and the reassignment between stop and sense codons.1,2 Stop codons are decoded by class I release factors (RFs).3 Whereas eukaryotes and archaea use a single RF to recognize all three stop codons,4,5 bacteria use two: RF1 is speciﬁc for UAA/UAG, and RF2 is speciﬁc for UAA/UGA.6 It is unknown why there are two class I RFs in bacteria while a single class I RF is suﬃcient for organisms from the other two domains. The process for stop codon reassignment and its potential association with RF evolution are also unclear. Natural code evolution occurs over millions of years. Extant organisms harboring altered genetic codes are at the end-point of the code evolution. There are no records of the initial causes of or responses to such altered genetic codes; further adaptations to and details of eventual ﬁxation are completely unknown. To enable in-depth investigation of code change and any concurrent cellular adaptations in real time, it is necessary to generate a model organism that is able to undergo such evolutionary processes in the laboratory. Synthetically recoding a genome may aﬀord new properties to the organism through encoding unnatural amino acids (Uaas) and preventing cross-contamination with wild type life forms.7 For successful genome recoding, the target codon must be reassigned to the new meaning in high eﬃciency and without ambiguity. An attractive route is to reassign the UAG stop codon to a Uaa in bacteria. Orthogonal tRNA/synthetase pairs have been engineered to incorporate Uaas into proteins in response to UAG,8−12 yet the presence of RF1 makes the meaning of UAG ambiguous, being a stop signal and a Uaa simultaneously. RF1 competition limits the incorporation of Uaas at a single UAG site with low eﬃciency; the addition of even a second UAG codon decreases protein yields precipitously.13 Although Uaas can be incorporated at more than one site into a protein,14,15 such low UAG-encoding eﬃciency prevents eﬀective use of Uaas at multiple sites to explore novel protein and organismal properties through directed evolution. In addition, the ambiguity of the UAG codon may hinder the eventual ﬁxation of an altered genetic code to an organism, because protein products truncated at the UAG sites can interfere with normal protein functions and have detrimental eﬀects on the host cell, thus preventing advanta- geous coding from being inherited and selected in directed evolution. To exclude Uaa incorporation at legitimate termination sites speciﬁed by UAG, endogenous UAG codons in the E. coli genome can be replaced with a synonymous UAA stop codon through genome engineering.16 However, for Received: May 9, 2012 Accepted: June 4, 2012 Published: June 5, 2012 © 2012 American Chemical Society 1337 dx.doi.org/10.1021/cb300229q | ACS Chem. Biol. 2012, 7, 1337−1344