ACS Chemical Biology sites (Supporting Figure S1). These results indicate that misreading of UAG in JX1.0 by endogenous tRNAs was outcompeted by the tRNATCyUrA/LW1RS pair, which speciﬁcally decodes UAG as pActF. To evaluate the initial response of E. coli to RF1 deletion and subsequent UAG reassignment, we assessed the health of JX1.0 using a growth assay. JX1.0 was healthy, cloneable, and stable in culture; no changes in phenotype or genotype were observed after growing over 200 generations. Compared to parental BL21(DE3), JX1.0 showed a slower doubling rate (Figure 4). Figure 4. RF1 knockout and UAG reassignment slow down the doubling of JX1.0. The growth curves for the RF1 knockout strain JX1.0 are represented with closed symbols and for the parental BL21(DE3) strain with open symbols. Squares represent cells only; circles represent cells transformed with the pAIO plasmid, which expresses the orthogonal tRNACTUyrA/LW1RS pair; and triangles represent cells transformed with the pAIO plasmid and grown in the presence of pActF. Error bars represent the s.e.m., n = 3. The doubling time for JX1.0 was 91 min compared to 26 min for BL21(DE3). This diﬀerence suggests that RF1 knockout creates burdens for cells due to the lack of proper termination and relatively weak missuppression of UAGs by near-cognate tRNAs, yet the eﬀect is not lethal as perceived before. Introduction of the pAIO plasmid expressing the orthogonal tRNATCyUrA/LW1RS pair in the absence of pActF increased the doubling of JX1.0 and BL21(DE3) to 135 and 41 min, respectively, possibly due to a general toxicity of expressing unacylated tRNAs. The addition of pActF to the growth medium together with the expression of tRNATCyUrA/LW1RS further increased the doubling time to 253 min for JX1.0, whereas BL21(DE3) was not aﬀected. This dramatic doubling reduction of JX1.0 is most likely from the eﬃcient incorporation of pActF at UAG positions throughout the proteome, reﬂecting the pressure generated by UAG reassign- ment to a sense codon. In contrast, BL21(DE3) expresses RF1, which competes at UAG positions for termination, thereby mitigating this pressure. We show here that RF1 is nonessential for the E. coli species, in contrast to the previous conclusion that RF1 is essential. By reverting Thr246 to Ala and removing the autoregulation of RF2 expression, we recently knocked out RF1 from an engineered E. coli strain MDS42 that has a reduced genome.13 Because ∼700 nonessential genes are deleted in MDS42 and multiple mutations are introduced into its RF2 gene, it is diﬃcult to conclude what factors speciﬁcally contribute to RF1 knockout and whether RF1 dispensability is a general property of E. coli. Moreover, MDS42 is not a wild type E. coli and not suitable for the investigation of code evolution and adaptation Letters since a variety of genes have been removed. By studying various nonengineered E. coli strains, we discovered here that a wild type RF2 is suﬃcient and necessary for RF1 removal without incurring compensatory mutations. As no other mutations are artiﬁcially introduced into these strains, our results clearly demonstrate that RF1 is nonessential for wild type E. coli. The successful RF1 knockout in multiple strains indicates that RF1 dispensability is a general property of E. coli without peculiar requirements. Unconditional RF1 removal can be explained by the usage of UAG in E. coli genome. UAG is the least-used stop codon in E. coli, terminating only ∼7% of the total genes.29 Among 302 essential genes in E. coli,44 only 7 genes are ended with the UAG codon, and these 7 genes all have either a UGA or UAA stop codon downstream in short distances (Supporting Figure S2). If the UAG is readthrough, this second diﬀerent stop codon can ensure the termination of these proteins with only a small number of amino acids appended. The additional amino acids may not completely disable the function of these proteins, allowing E. coli to survive as observed in this study. The few UAG-ending essential genes and the presence of double stop codons can account for the nonessentiality of RF1. Consistently, we show here that a wild type RF2 but not the weakened A246T mutant permitted RF1 knockout, suggesting that eﬃcient termination of the dominant UAA and UGA stop codons is required for E. coli survival. Knockout of RF1 provides novel insights on the evolution of RFs and the genetic code. A major distinction of bacteria from eukaryotes and archaea in protein translation is that bacteria use two diﬀerent RFs to recognize the stop codons, whereas a single RF is suﬃcient for organisms in other domains. This diﬀerence has served as evidence that the evolution of RF and translation termination between bacteria and eukaryotes/ archaea is nonconserved.45,46 Our results show that an autonomous E. coli strain with a single class I RF can be generated. The viability of such a bacterium blurs the apparent two-vs-one RF distinction between prokaryotes and eukar- yotes/archaea, suggesting that RF evolution in the three domains of life might be more similar than current diﬀerences suggest. In addition, certain eukaryotes have been found to reassign a stop codon to a sense codon and restrict the recognition of eRF1 to the rest of the stop codons.2,17,18 It is unclear whether stop codon reassignment caused eRF1 restriction or vice versa during the code evolution. We show here that bacteria, in addition to eukaryotes found in nature, can also undergo such stop codon reassignment accompanied by RF changes. Moreover, our results demonstrate that RF changes can synthetically drive the reassignment of a stop codon to an amino acid, providing experimental evidence for this hypothetic evolution pathway. An autonomous RF1 knockout bacterium will enable new research for understanding code evolution. A major challenge in studying the evolution of the genetic code is that many questions are out of reach of direct experimentation.47 No organisms exist containing a primitive or intermediate genetic code for comparison, and natural code evolution takes millions of years. The RF1 knockout strain reported here now aﬀord a previously unavailable model organism to study otherwise intractable questions on the codon reassignment process in real time. We show here that RF1 knockout and the introduction of an orthogonal tRNA/synthetase pair completely reassigned UAG from a stop signal to an amino acid, setting an initial stage for a bacterium to adopt this altered genetic code. Such a stage 1341 dx.doi.org/10.1021/cb300229q | ACS Chem. Biol. 2012, 7, 1337−1344