ACS Chemical Biology Letters Figure 3. UAG-decoding orthogonal tRNA/synthetase pair reassigns the UAG codon to an amino acid in JX1.0. (A) The plasmid pAIO-EGFP(n- TAG) encodes an orthogonal tRNATCyUrA/LW1RS pair and the EGFP reporter with various numbers of TAG codons. Structure of pActF is shown to the right. (B) Western blot analysis of EGFP expression by pAIO-EGFP(n-TAG) in BL21(DE3) and JX1.0 cells in the presence or absence of pActF. A penta-His antibody was used to detect the Hisx6 tag fused at the N-terminus of EGFP. (C) In-cell ﬂuorescence intensity of EGFP expressed by pAIO-EGFP(n-TAG) in BL21(DE3) and JX1.0 cells in the presence or absence of pActF. Samples were normalized for cell numbers in each lane. Measurement was performed on three independent batches of cells, and error bars represent the SEM. (D) SDS-PAGE analysis of EGFP proteins puriﬁed from JX1.0 cells expressing pAIO-EGFP(n-TAG) in the absence or presence of pActF. (E) Fourier transform mass spectrometric analysis of EGFP expressed by pAIO-EGFP(3-TAG) in JX1.0 with pActF in the growth medium. The spectra show the intact precursor ions (charge deconvoluted) for the tryptic fragments of EGFP. Monoisotopic masses measured with high accuracy indicate that pActF was incorporated at all three UAG sites. Peptide sequences are shown, and X represents the UAG encoded site. Top: site 39, expected [M + H]+ =1529.65 Da, measured 1529.66 Da. Middle: site 151, expected [M + H]+ =1999.89 Da, measured 1999.90 Da; the 2015.89 peak corresponds to Met oxidation of this pActF-containing peptide. Bottom: site 182, expected [M + H]+ =4498.18 Da, measured 4498.14 Da. No peaks corresponding to any other amino acids at these UAG positions were detected. The signal-to-noise ratio is >1000, translating to a ﬁdelity for pActF incorporation of >99.9%. Tandem MS spectra of these peptides are shown in Supporting Figure S1, which unambiguously conﬁrm the peptide sequences and pActF at position X. speciﬁc LW1RS is less active than the wild type synthetases39,40 and thus may generate fewer aminoacylated orthogonal tRNAs. Second, the orthogonal tRNATCyUrA has not been evolutionarily optimized for UAG decoding, whereas many natural tRNAs are posttranscriptionally modiﬁed through evolution for eﬃcient codon recognition.41 Third, natural aminoacyl-tRNAs have also been ﬁne-tuned for binding to the elongation factor Tu and the ribosome to achieve eﬃcient translation,42,43 whereas the pActF-charged orthogonal tRNA has not been optimized for binding to either. All of these factors can lead to less eﬃcient decoding of UAG as pActF and ribosome drop-oﬀ during translation, resulting in truncated protein products. The less eﬃcient decoding of UAG to pActF can also have accumulative eﬀect to account for the decrease of protein yield for the 10- TAG mutant. Nonetheless, the underlying mechanism for generating these truncated products in the absence of RF1 is intriguing and warrants further studies. To identify the amino acid incorporated in response to UAG in JX1.0, we puriﬁed EGFP expressed in JX1.0 using pAIO- EGFP(3-TAG) in the presence of pActF and analyzed it with mass spectrometry (Figure 3E). The monoisotopic masses of the tryptic peptides clearly showed that pActF was incorporated at all three UAG sites. No peaks corresponding to the incorporation of other amino acids at the UAG sites were detected. The precursor ions of the peptides containing the UAG sites were individually fragmented with an ion trap mass spectrometer. The fragment ion masses were unambiguously assigned, conﬁrming that pActF was incorporated at the UAG 1340 dx.doi.org/10.1021/cb300229q | ACS Chem. Biol. 2012, 7, 1337−1344