Expanding the Genetic Code of Yeast A R T I C L E S Figure 4. Characterization of unnatural amino acid incorporation in yeast with the orthogonal MbPylRS/MmtDNAPCyUlA pair. (A) Amber suppression efﬁciency of hSOD33TAG-His6 in yeast in the presence or absence of 1 (5 mM), 2 (10 mM), 3 (10 mM), 4 (2 mM), or 5 (1.3 mM) by anti-His6 western blot. Yeast cells containing the hSOD expression construct were transformed with the dicistronic SctDNAUACrgU-MmtDNACPyUlA construct for expressing the orthogonal MmtDNAPyl in yeast and the appropriate aminoacyl-tRNA synthetase (aaRS). PylRS (wild-type MbPylRS), AcKRS (a variant of MbPylRS that has been CUA evolved to use 23), TfaKRS (a variant of MbPylRS that can use 3, see text), PcKRS (a variant of MbPylRS that has been evolved to use 42). (B) Coomassie SDS-PAGE analysis of puriﬁed hSOD from expressions in the presence and absence of 1, 2, or 3. Full protein MS (C-E) and Glu-C MS/MS (F-H) conﬁrm the incorporation of unnatural amino acids 1 (C/F found 16691 ( 1.5 Da, expected 16691 Da), 2 (D/G found 16651 ( 1.5 Da, expected 16651) and 3 (E/H found 16705 ( 1.5 Da, expected 16705) at the genetically encoded site. hSOD is copuriﬁed as a heterodimer with yeast SOD (minor additional peak in spectra at 15722 Da; identity was conﬁrmed by Glu-C MS/MS). For full gels and western blots and larger versions of MS and MS/MS data see Supporting Information Figure 2. in yeast proteins.24 Amino acid 2 may be used for producing acetylated proteins directly in yeast and synthetically controlling processes normally regulated by acetylation in yeast. Amino acid 3 is a very poor substrate for sirtuins, but not for HDACs,25 and should allow us to install irreversible acetylation at sites directly regulated by sirtuins in ViVo. It should allow us to probe the deacetylases that act on a given site in a protein. Amino acid 4 is a photocaged lysine with demonstrated utility for controlling protein function in eukaryotic cells,2 and we anticipate that genetically encoded photocontrol of proteins in yeast will be a powerful approach for gaining a temporal and spatial understanding of cellular processes. Amino acid 5 is a photo-cross-linking amino acid with demonstrated utility for mapping protein interactions in E. coli,23 and we believe that (24) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004. (b) Meldal, M.; Tornøe, C. W. Chem. ReV. 2008, 108, 2952. (25) (a) Smith, B. C.; Denu, J. M. J. Biol. Chem. 2007, 282, 37256. (b) Smith, B. C.; Hallows, W. C.; Denu, J. M. Chem. Biol. 2008, 15, 1002. this will ﬁnd wide utility in mapping protein-protein interac- tions in yeast. Given the growing list of amino acids that can be incorporated using MbPylRS and its variants,1-6 we antici- pate that our approach will allow the introduction of a wide range of chemical functional groups into yeast. Finally, the strategies we have explored for creating and expressing heter- ologous, orthogonal tRNAs in yeast may be useful for improving other orthogonal aminoacyl-tRNA synthetase/tRNACUA pairs.8-10 Experimental Section General Methods. Nε-[(2-Propynyloxy)carbonyl]-L-lysine,5 Nε- [(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl]-L-lysine2 and Nε-[(2-(3-methyl-3H-diazirin-3-yl)ethoxy)carbonyl]-L-lysine23 were synthesized as previously reported. Nε-Acetyl-L-lysine and Nε- triﬂuoroacetyl-L-lysine were purchased from Bachem. Northern Blot Analysis. Total RNA was puriﬁed from yeast cells using TRI reagent (Sigma) and ethanol precipitated. The RNA was denatured, separated on a 6% Novex TBE-urea gel (Invitrogen), blotted onto Biodyne B modiﬁed nylon membrane (Thermo Scientiﬁc), and cross-linked by UV ﬁxation. The membrane was hybridized overnight at 55°C with a biotinylated probe 5′- J. AM. CHEM. SOC. 9 VOL. 132, NO. 42, 2010 14823