Expanding the Genetic Code of Yeast Figure 2. Creating a functional tRNACPyUlA in yeast. (A) The consensus A- and B-box sequences and the A- and B-box sequences of MbtDNAPyl . (B) The MbtDNACPyUlA expression constructs created and examined in CtUhAis work. Constructs 6a-d were created using the 5′ and 3′ ﬂanks from distinct tRNAs as described in the text. (C) Northern blots for MbtDNAPyl CUA expression from various constructs. (D) Phenotyping constructs for amber suppression in MaV203:pGADGAL4(2TAG) cells, where 3AT is 3-ami- notriazole and 1 was used at 2 mM. Cells contained MbPylRS and the appropriate MbtDNAPyl expression construct. CUA produce mature SNR52 snoRNA.12 A previous report suggested that adding 5′-SNR52 and 3′-SUP4 ﬂanking sequences to EctDNATyr and EctDNALCeUuA enhanced their amber suppression CUA in yeast.13 When MbtRNAPyl CUA was cloned between 5′-SNR52 and 3′-SUP4 ﬂanking sequences (Figure 2B construct 5), we could detect weak MbtRNACPyUlA transcription by northern blot (Figure 2C), and when the cassette was transformed into MaV203:pGADGAL4(2TAG) containing MbPylRS and grown in the presence of 1, we observed blue coloration on X-Gal plates but not growth in the absence of histidine in the presence of 40 mM 3-aminotriazole (3AT) (Figure 2D). These data suggest that addition of extragenic A- and B-box sequences via the 5′-SNR52 and 3′-SUP4 ﬂanking sequences can partially compensate for the absence of consensus A- and B-box sequences in MbtRNACPyUlA. However, since the EcTyrRS/ tRNACTyUrA orthogonal pair supports growth on media lacking histidine and containing 40 mM 3AT8,9 but this system does not, we decided that the system was suboptimal and opted to explore further extragenic sequences. (12) Guffanti, E.; Ferrari, R.; Preti, M.; Forloni, M.; Harismendy, O.; Lefebvre, O.; Dieci, G. J. Biol. Chem. 2006, 281, 23945. (13) Wang, Q.; Wang, L. J. Am. Chem. Soc. 2008, 130, 6066. A R T I C L E S The yeast U6 (SNR6) gene assembles the same RNA polymerase III transcriptional machinery as tRNA genes but possesses an additional TATA-box promoter element 30 base pairs upstream of the transcription start site that binds TFIIIB.14 The TATA-box enables TFIIIC-independent RNA polymerase III recruitment and is proposed to overcome the large separation (240 bp) of the A- and B-box promoter elements of this gene.15 Several yeast tRNAs, some of which contain large introns between the A- and B-boxes, have TATA boxes that allow TFIIIC-independent RNA polymerase transcription.15 We rea- soned that by incorporating the ﬂanking sequences of these genes into our tRNA cassettes it may be possible to compensate for the poor A- and B-box consensus of MbtRNACPyUlA. We created constructs where the 5′-ﬂanking region of SNR6, Ile{TAT}LR1, Pro{TGG}FL, and Asp{GTC}KR and the 3′- ﬂanking region of SNR6 sandwich MbtRNACPyUlA (Figure 2B constructs 6a-d). We also added a consensus sequence16 found at the transcription start site of yeast tRNAs to the SNR6 construct. Northern blots revealed low-level tRNA production from construct 6a (Supporting Information Figure 1). However, we did not observe phenotypes consistent with amber suppres- sion when any of these constructs were transformed into MaV203:pGADGAL4(2TAG) containing MbPylRS and grown in the presence of 1 (Figure 2D). These data suggested that, while these promoter elements may compensate for increases in the A- and B-box spacing, they cannot efﬁciently compensate for defects in the A- and B-box sequence in MbtRNACPyUlA. Yeast possess an unusual dicistronic tDNAUACrgU-tDNAGAUspC gene in which the two mature tRNAs are generated from a single precursor RNA.17 The A- and B-box promoter in the tDNAUACrgU gene directs the transcription of the precursor, and the transcrip- tion of tDNAAGsUpC is entirely dependent on the promoter elements of tDNAUACrgU. This suggests that tDNAUACrgU may provide the A- and B-box sequences required to transcribe tDNAs inserted in place of tDNAGAUspC.18 Indeed replacing tDNAGAUspC with human initiator tDNA or a transcriptionally inactive yeast tyrosine suppressor tDNA allows these tDNAs to be transcribed and processed to produce functional tRNAs in yeast.19 To test this system for the transcription of MbtRNACPyUlA, we constructed a SctDNAAUrCgU-MbtDNAPCyUlA cassette containing the natural 5′-, 3′-, and 10-base pair linker sequences (Figure 2B construct 7). Northern blot analysis revealed that MbtRNACPyUlA was transcribed from this construct much more efﬁciently than any other construct tested (Figure 2C). When transformed into MaV203:pGADGAL4(2TAG) in the presence of MbPylRS and 1, the SctDNAAUrCgU-MbtDNAPCyUlA cassette conferred survival on media lacking histidine and containing 40 mM 3AT, and produced the strongest blue color of any construct tested when incubated with X-Gal (Figure 2D). The tRNA constructs we discovered that are both transcribed (as judged by northern blot) and functional (as judged by phenotyping (constructs 5 and 7)) showed amber suppression (14) Brow, D. A.; Guthrie, C. Genes DeV. 1990, 4, 1345. (15) Dieci, G.; Percudani, R.; Giuliodori, S.; Bottarelli, L.; Ottonello, S. J. Mol. Biol. 2000, 299, 601. (16) Raymond, K. C.; Raymond, G. J.; Johnson, J. D. EMBO J. 1985, 4, 2649. (17) Schmidt, O.; Mao, J.; Ogden, R.; Beckmann, J.; Sakano, H.; Abelson, J.; So¨ll, D. Nature 1980, 287, 750. (18) (a) Kjellin-Straby, K.; Engelke, D. R.; Abelson, J. DNA 1984, 3, 167. (b) Reyes, V. M.; Newman, A.; Abelson, J. Mol. Cell. Biol. 1986, 6, 2436. (19) (a) Francis, M. A.; Rajbhandary, U. L. Mol. Cell. Biol. 1990, 10, 4486. (b) Stråby, K. B. Nucleic Acids Res. 1988, 16, 2841. J. AM. CHEM. SOC. 9 VOL. 132, NO. 42, 2010 14821