ACS Chemical Biology expression in BL21(DE3) and the RF1 knockout strain JX1.0 (Figure 2A). As expected, BL21(DE3) cells expressing either Figure 2. UAG codon in the RF1 knockout strain JX1.0 is suppressed by endogenous tRNAs with mispairing anticodons. (A) An EGFP gene with the TAG codon at permissive sites was driven by the arabinose promoter in the plasmid pBAD-EGFP(n-TAG) to test EGFP expression. (B) In-cell ﬂuorescence intensity measured with ﬂuorometry for BL21(DE3) and JX1.0 cells transformed with pBAD-EGFP(1-TAG) or pBAD-EGFP(3-TAG). Samples were normalized for cell numbers. (C) Fourier transform mass spectro- metric analysis of EGFP protein puriﬁed from JX1.0 expressing pBAD- EGFP(1-TAG). The spectrum shows the intact precursor ions (charge deconvoluted) for the tryptic fragment of EGFP (HNIEDGSVQ- LADHXQQNTPIGDGPVLLPDNHYLSTQSALSK, X representing the UAG encoded site). Monoisotopic masses measured with high accuracy indicate that Gln, Tyr, and Trp (expected [M + H]+ = 4437.17, 4472.17, and 4495.19 Da; measured 4437.16, 4472.16, and 4494.16 Da, respectively) were incorporated at the UAG site. No other amino acids were detected at the UAG site. the 1- or 3-TAG reporter showed no EGFP ﬂuorescence, indicating that RF1 terminates EGFP translation at UAG normally. Surprisingly, JX1.0 showed a high level of ﬂuorescence from the 1-TAG EGFP mutant and a small but reproducible level of ﬂuorescence from the 3-TAG EGFP mutant (Figure 2B). These data suggest that some endogenous tRNAs can suppress the UAG codon in JX1.0. To reveal the identity of these tRNAs, we puriﬁed EGFP protein expressed with the 1-TAG reporter in JX1.0 and identiﬁed the amino acid incorporated at the UAG site using mass spectrometry (Figure 2C). Tyr, Gln, and Trp were found at the UAG site through Fourier transform mass spectrometric analysis of the tryptic fragments of EGFP. To ensure that this is not from a suppressor mutation in the endogenous tRNAs, chromosomal tRNATyr, tRNAGln, and tRNATrp were rese- quenced, and all were conﬁrmed to be wild type in JX1.0. The Letters anticodons of these three tRNAs have only a single base mispairing with UAG, so these tRNAs can weakly misread the UAG codon.37 Misreading of UAG by these tRNAs was not obvious in the presence of RF1 in BL21(DE3) but became marked in JX1.0 after RF1 removal. The unconditional knockout of RF1 opens up the possibility of reassigning the meaning of the UAG codon completely, so that the UAG codon does not ambiguously encode a stop signal and an amino acid simultaneously. We attempted to recruit UAG for an amino acid in JX1.0, so as to mimic a possible code evolution pathway implicated by RF constriction in stop codon recognition in Tetrahymena, Euplotes, and other eukaryotes.17,18 An orthogonal tRNA/synthetase pair, the tRNATCyUrA/LW1RS pair, was introduced into JX1.0. This pair does not crosstalk with endogenous E. coli tRNA/synthetase pairs and functionally couples with E. coli’s protein translational machinery.8,38 The tRNATCyUrA decodes the UAG codon speciﬁcally through its anticodon CUA, and LW1RS is engineered to charge the tRNATCyUrA with the Uaa p-acetyl-L-phenylalanine (pActF).39 An EGFP gene containing 1-, 2-, 3-, or 10-TAG codons was co- expressed with tRNATCyUrA/LW1RS in a single plasmid pAIO- EGFP(n-TAG) (Figure 3A). EGFP expression was assayed by Western blotting (Figure 3B) and in-cell ﬂuorescence (Figure 3C). When pActF was not added to the growth media, BL21(DE3) cells expressed a small amount of full-length EGFP with the 1-TAG reporter only, suggesting that the tRNATCyUrA/LW1RS pair incorporates a natural amino acid in very low eﬃciency in the absence of the cognate pActF. No full-length EGFP was detected for the 2-, 3-, or 10-TAG reporters. In contrast, JX1.0 expressed full-length EGFP for 1-, 2-, 3-, and 10-TAG mutants, although the eﬃciency decreased with the number of UAG codons. We then puriﬁed the EGFP protein from JX1.0 expressing pAIO- EGFP(1-TAG) in the absence of pActF (Figure 3D) and analyzed it with mass spectrometry. Consistently, Tyr, Gln, and Trp were found at the UAG site as observed in Figure 2C, conﬁrming misreading by endogenous tRNAs in JX1.0. When pActF was supplied in the growth medium, BL21- (DE3) cells showed eﬃcient expression of the 1-TAG EGFP mutant, but EGFP expression decreased precipitously with the addition of each UAG codon due to the competition from RF1 termination. The use of 3 UAG codons in EGFP virtually abolished protein expression, and no protein could be detected at all in the 10-TAG mutant. In stark contrast, the RF1-deletion strain JX1.0 showed high expression of EGFP in all mutants in the presence of pActF, as indicated by Western (Figure 3B) and in-cell ﬂuorescence (Figure 3C). EGFP proteins were puriﬁed with yields of 8.5 (±0.4), 7.1 (±0.4), 9.7 (±0.3), and 1.2 (±0.1) mg/L for the 1-, 2-, 3-, and 10-TAG EGFP samples, respectively (Figure 3D). There was no decrease in incorporation eﬃciency when the UAG codon was increased from 1 to 2 or 3, indicating that UAG is changed to a sense codon in JX1.0. The drop oﬀ in yield in the 10-TAG EGFP sample is likely because 10 pActF interferes with EGFP folding and/or stability, which is corroborated with the lack of ﬂuorescence from 10-pActF EGFP (Figure 3C). Although JX1.0 had no RF1 to terminate protein translation at the UAG codon, a very small amount of protein was detected on the Western blot but could not be detected with mass spectrometry that seemed to be truncated at the UAG site (Figure 3B). We think that the decoding of the UAG codon as the Uaa pActF may not be as eﬃcient as the decoding of canonical sense codons as natural amino acids. First, the pActF- 1339 dx.doi.org/10.1021/cb300229q | ACS Chem. Biol. 2012, 7, 1337−1344