ACS Chemical Biology complete reassignment of UAG to a sense codon, a necessary and critical step is to knock out RF1 from the E. coli genome. In some eukaryotes such as ciliates and green algae, the reassignment of a stop codon to a sense codon is accompanied by convergent changes in eRF1.2 For instance, the eRF1 of Tetrahymena restricts its recognition to UGA, and UAA/UAG are reassigned to Gln; the eRF1 of Euplotes recognizes only UAA/UAG as stop codons, and UGA is used to encode Cys.17,18 In bacteria, Mycoplasma species have lost the RF2 gene, and the UGA codon encodes Trp instead.19 However, Mycoplasma are obligatory pathogens with highly reduced genomes. To date, no free-living bacterium has been found lacking either RF1 or RF2.19 For E. coli, RF1 has been reported to be essential,20 and only conditionally lethal knockouts have been described.21,22 Recently we managed to knock out RF1 from a special E. coli strain that has a reduced genome and a mutated RF2 gene.13 Herein we discovered that the dispensability of RF1 is a general property of wild type E. coli, arguing against the paradigm that RF1 is essential. We revealed the underlying mechanism for preventing RF1 knockout, and generated autonomous RF1 deletion strains valuable for studying code evolution and for genome recoding. We attempted to replace the RF1-encoding gene, prfA, with the chloramphenicol acetyltransferase gene in a variety of E. coli strains (Figure 1) using the established λ red recombinase- based homologous recombination method.23 E. coli K-12 and B are the two progenitors from which most E. coli strains are derived.24 Reported attempts to knockout RF1 have used only E. coli K-12 strains and were unsuccessful.20,25 We initially attempted to delete RF1 in the three common K-12 strains MG1655, DH10β, and HT115. Knockout of RF1 was assayed by genomic PCR amplifying the prfA locus (Figure 1A). Consistent with previous reports, we could not knock out RF1 in any of these K-12 strains (Figure 1B). We next attempted the RF1 knockout with two other K-12 strains that contain alterations related to translational termination. The BP5α strain harbors a glutaminyl amber suppressor tRNA, making the UAG coding ambiguous for either a stop signal or Gln. This strain would test if stop codon ambiguity is a factor to prime RF1 removal. The MDS42 strain has nearly 700 nonessential genes deleted26 and was used to assess if a reduced genome size and termination load could assist with RF1 removal. However, no RF1 knockouts were generated from these two strains either, suggesting that amber suppression and a minimal genome are insuﬃcient for RF1 removal in K-12 derivatives. All K-12 strains contain a peculiar A246T mutation in the RF2-encoding gene prf B, lowering the release activity of RF2 for UAA 5-fold.27,28 The UAA codon accounts for the termination of ∼64% of E. coli genes.29 We reasoned that the A246T mutation might severely impair the ability of RF2 to recognize all UAA stop codons upon removal of RF1 and thus prevent the RF1 knockout. We therefore tested three common E. coli B strains because B strains encode wild type RF2 that contains Ala246.28 Indeed, RF1 knockout was successful with all three B strains tested; REL606, BL21, and BL21(DE3) were used to generate the knockout strains CW1.0, CW2.0, and JX1.0, respectively (Figure 1A). In addition, to determine if the A246T mutation in RF2 prevents RF1 removal in K-12 strains, we reverted the A246T mutation to Ala in the K-12 strain DH10β to generate the DH10βf strain. This strain also permitted the direct knockout of RF1 (Figure 1B). These Letters Figure 1. RF1 can be knocked out from E. coli strains containing wild type RF2. (A) The RF1-encoding gene prfA was replaced with the chloramphenicol acetyltransferase (cat) gene through homologous recombination using the phage λ red recombinase expressed with plasmid pKD46.23 Chloramphenicol-resistant colonies were screened using PCR for cat replacement and conﬁrmed with sequencing. The agarose gels show the PCR results for parental and knockout strains as labeled. (B) A list of E. coli strains tested for RF1 knockout. K-12 and B strains are the most widely used E. coli and are the subjects of classical experiments. Among other genomic diﬀerences between K-12 and B strains,24 A246T in RF2 is a peculiar mutation in K-12 strains that reduces the release activity of RF2 for the stop codon UAA. This mutation was reverted to wild type Ala in DH10βf. The glnV44 gene encodes a glutaminyl amber suppressor tRNA. RF1 was successfully knocked out in three B strains and the DH10βf strain. results indicate that RF1 is not essential in E. coli, in contrast to the previous conclusions.20,25 Full genomic sequencing was performed on RF1 knockout strains JX1.0, CW1.0, and CW2.0 and compared to their respective parental strains.30,31 The RF1 deletion was veriﬁed in all cases. For CW1.0 and CW2.0, no other mutations were found throughout the genome. For JX1.0, only seven additional single nucleotide polymorphisms (SNPs) were found (Support- ing Table S1). Six of these SNPs are silent mutations in two genes of phage origin. None of the SNPs correspond to known mutations that complement an RF1 deﬁciency.32−35 These results indicate that RF1 was knocked out from the parental E. coli strains without incurring compensatory mutations. What would happen to the UAG site during protein translation in the absence of RF1? We mutated one and three tyrosine codons to TAG in the enhanced green ﬂuorescent protein (EGFP) gene36 to generate the 1-TAG and 3-TAG EGFP reporters, respectively, and tested their 1338 dx.doi.org/10.1021/cb300229q | ACS Chem. Biol. 2012, 7, 1337−1344