Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 132 (42), 14819-24

Expanding the Genetic Code of Yeast for Incorporation of Diverse Unnatural Amino Acids via a pyrrolysyl-tRNA synthetase/tRNA Pair

Affiliations

Expanding the Genetic Code of Yeast for Incorporation of Diverse Unnatural Amino Acids via a pyrrolysyl-tRNA synthetase/tRNA Pair

Susan M Hancock et al. J Am Chem Soc.

Abstract

We report the discovery of a simple system through which variant pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pairs created in Escherichia coli can be used to expand the genetic code of Saccharomyces cerevisiae. In the process we have solved the key challenges of producing a functional tRNA(CUA Pyl) in yeast and discovered a pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pair that is orthogonal in yeast. Using our approach we have incorporated an alkyne-containing amino acid for click chemistry, an important post-translationally modified amino acid and one of its analogs, a photocaged amino acid and a photo-cross-linking amino acid into proteins in yeast. Extensions of our approach will allow the growing list of useful amino acids that have been incorporated in E. coli with variant pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pairs to be site-specifically incorporated into proteins in yeast.

Figures

Figure 1
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.
Figure 2
Figure 2
Creating a functional tRNACUAPyl in yeast. (A) The consensus A- and B-box sequences and the A- and B-box sequences of MbtDNACUAPyl. (B) The MbtDNACUAPyl expression constructs created and examined in this work. Constructs 6a−d were created using the 5′ and 3′ flanks from distinct tRNAs as described in the text. (C) Northern blots for MbtDNACUAPyl expression from various constructs. (D) Phenotyping constructs for amber suppression in MaV203:pGADGAL4(2TAG) cells, where 3AT is 3-aminotriazole and 1 was used at 2 mM. Cells contained MbPylRS and the appropriate MbtDNACUAPyl expression construct.
Figure 3
Figure 3
MmtDNACUAPyl is orthogonal in yeast, but MbtDNACUAPyl directs the incorporation of alanine and is not orthogonal in yeast. (A) Constructs used to compare orthogonality of tRNACUAPyl in yeast. (B) Analysis of amber suppression by expression of hSOD33TAG-His6 and detection by anti-His6 western blot. Yeast cells containing the hSOD expression plasmid, MbPylRS and the dicistronic SctDNAUCUArg−tDNACUAPyl construct were grown in the presence or absence of 1 (5 mM). (C) ESI-MS shows that alanine is incorporated into hSOD33TAG in cells producing amber suppressor MbtDNACUAPyl from construct 7 (Found 16553 ± 1.5 Da, expected 16553 Da), confirming that MbtDNACUAPyl is a substrate for yeast alanyl-tRNA synthetases.
Figure 4
Figure 4
Characterization of unnatural amino acid incorporation in yeast with the orthogonal MbPylRS/MmtDNACUAPyl 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 SctDNAUCUArgMmtDNACUAPyl construct for expressing the orthogonal MmtDNACUAPyl in yeast and the appropriate aminoacyl-tRNA synthetase (aaRS). PylRS (wild-type MbPylRS), AcKRS (a variant of MbPylRS that has been evolved to use 2(3)), TfaKRS (a variant of MbPylRS that can use 3, see text), PcKRS (a variant of MbPylRS that has been evolved to use 4(2)). (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.

Similar articles

See all similar articles

Cited by 59 PubMed Central articles

See all "Cited by" articles

References

    1. Neumann H.; Peak-Chew S. Y.; Chin J. W. Nat. Chem. Biol. 2008, 4, 232. - PubMed
    1. Gautier A.; Nguyen D. P.; Lusic H.; An W.; Deiters A.; Chin J. W. J. Am. Chem. Soc. 2010, 132, 4086. - PubMed
    1. Neumann H.; Hancock S. M.; Buning R.; Routh A.; Chapman L.; Somers J.; Owen-Hughes T.; van Noort J.; Rhodes D.; Chin J. W. Mol. Cell 2009, 36, 153. - PMC - PubMed
    1. Zhao S.; Xu W.; Jiang W.; Yu W.; Lin Y.; Zhang T.; Yao J.; Zhou L.; Zeng Y.; Li H.; Li Y.; Shi J.; An W.; Hancock S. M.; He F.; Qin L.; Chin J.; Yang P.; Chen X.; Lei Q.; Xiong Y.; Guan K. L. Science 2010, 327, 1000. - PMC - PubMed
    2. Nguyen D. P.; Garcia Alai M. M.; Kapadnis P. B.; Neumann H.; Chin J. W. J. Am. Chem. Soc. 2009, 131, 14194. - PubMed
    3. Fekner T.; Li X.; Lee M. M.; Chan M. K. Angew. Chem., Int. Ed. 2009, 48, 1633. - PubMed
    4. Li W. T.; Mahapatra A.; Longstaff D. G.; Bechtel J.; Zhao G.; Kang P. T.; Chan M. K.; Krzycki J. A. J. Mol. Biol. 2009, 385, 1156. - PubMed
    5. Li X.; Fekner T.; Ottesen J. J.; Chan M. K. Angew. Chem., Int. Ed. 2009, 48, 9184. - PubMed
    6. Yanagisawa T.; Ishii R.; Fukunaga R.; Kobayashi T.; Sakamoto K.; Yokoyama S. Chem. Biol. 2008, 15, 1187. - PubMed
    1. Nguyen D. P.; Lusic H.; Neumann H.; Kapadnis P. B.; Deiters A.; Chin J. W. J. Am. Chem. Soc. 2009, 131, 8720. - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback