A R T I C L E S Hancock et al. Figure 1. Genetically encoded incorporation of new unnatural amino acids in yeast. (A) Unnatural amino acids used in this study. (B) Amber suppression by foreign tRNAs in yeast. (a) The tRNA gene is transcribed by RNA polymerase III using A- and B-box promoter sequences internal to the structural gene. (b) Processing of tRNA precursor involving cleavage of 5′ and 3′ ends and addition of 3′-CCA. (c) Export to the cytoplasm for aminoacylation by aminoacyl- tRNA synthetases with an unnatural amino acid. (d) Ribosome-mediated incorporation of the unnatural amino acid in response to an amber codon on the mRNA. (e) Production of a full length protein containing an unnatural amino acid at the genetically deﬁned site. and demonstrate the incorporation of several useful unnatural amino acids using variants of this pair created in E. coli (Figure 1). Results and Discussion To investigate the amber suppressor activity and potential orthogonality of the MbPylRS/tRNACPyUlA pair in S. cereVisiae we used MaV203:pGADGAL4(2TAG) cells.8,9 This yeast strain contains a GAL4 transcriptional activator gene bearing amber codons, is auxotrophic for histidine, and contains HIS3 and LacZ genes on GAL4-activated promoters. When a functional amber suppression system, such as the EcTyrRS/tRNACTyUrA pair,8,9 is transformed into this strain, full length GAL4 is produced, leading to activation of LacZ and HIS3 genes. Transcription of these genes allows cells to grow in the absence of histidine and turn blue in the presence of X-Gal. We replaced the functional EcTyrRS/tRNACTyUrA pair with the MbPylRS/MbtRNACPyUlA pair (Figure 2B construct 1) and sup- plemented with Nε-[(2-propynyloxy)carbonyl]-L-lysine (1) (Fig- ure 1A, a known substrate for MbPylRS5) in MaV203: pGADGAL4(2TAG). These cells were unable to grow in media lacking histidine and did not turn blue in the presence of X-Gal, suggesting that this original construct is not functional (Figure 2D). We demonstrated by western blot that the yeast codon- optimized MbPylRS was expressed in yeast cells (data not shown). However, analysis of northern blots indicated that MbtRNACPyUlA was not transcribed from our initial construct (Figure 2C). Since the EctRNATCyUrA gene contains the consensus A- and B-box RNA polymerase III promoter sequences that direct its transcription in yeast,11 but MbtRNACPyUlA does not (11) Edwards, H.; Schimmel, P. Mol. Cell. Biol. 1990, 10, 1633. 14820 J. AM. CHEM. SOC. 9 VOL. 132, NO. 42, 2010 (Figure 2A), it seemed likely that additional promoter elements would be required to direct the transcription of MbtRNACPyUlA. To address the challenge of creating new promoter elements to direct the transcription of MbtRNACPyUlA, we investigated strategies to introduce A- and B-box sequences into our tRNA expression construct. We ﬁrst mutated the sequence of the MbtRNACPyUlA gene to contain either near-consensus A box sequences (A11C/U15G/T24G, Figure 2B construct 2) or B box sequences (A56C, Figure 2B construct 3). Northern blot analysis demonstrated that the A56C mutation in the B box, led to very low but detectable levels of the mutant MbtRNAPCyUlA (Supporting Information Figure 1), while expression of the (A11C/U15G/ T24G) mutant tRNA was not detectable by northern blot. However, when the A56C mutant of MbtRNAPCyUlA and MbPylRS were transferred to MaV203:pGADGAL4(2TAG) in the pres- ence of 1, we did not observe phenotypes consistent with amber suppression (Figure 2D). This implies that either the tRNA is transcribed but not correctly folded or processed, or that the mutation abolishes synthetase recognition. Combining the A- and B-box mutations (Figure 2B construct 4) led to low levels of detectable tRNA production (Supporting Information Figure 1), but did not give phenotypes consistent with amber suppression (Figure 2D). Since enhancing the transcription of MbtRNAPCyUlA by mutation of the A- and B-box sequences within the structural gene did not produce a functional amber suppressor, we next investigated the potential of constructs that might augment the transcription of MbtRNACPyUlA using extragenic sequences. The 5′-leader sequence of the yeast SNR52 primary transcript contains A- and B-box promoters that are post-transcriptionally removed to