Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Proc Natl Acad Sci U S A
2006 Jun 06;10323:8650-5. doi: 10.1073/pnas.0510817103.
Show Gene links
Show Anatomy links
In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression.
Rodriguez EA
,
Lester HA
,
Dougherty DA
.
???displayArticle.abstract???
Site-specific incorporation of unnatural amino acids (UAAs) into proteins is a valuable tool for studying structure-function relationships, incorporating biophysical probes, and elucidating protein-protein interactions. In higher eukaryotic cells, the methodology is currently limited to incorporation of a single UAA in response to a stop codon, which is known as nonsense suppression. Frameshift suppression is a unique methodology for incorporating UAAs in response to quadruplet codons, but currently, it is mostly limited to in vitro protein translation systems. Here, we evaluate the viability of frameshift suppression in Xenopus oocytes. We demonstrate UAA incorporation by using yeast phenylalanine frameshift suppressor (YFFS) tRNAs that recognize two different quadruplet codons (CGGG and GGGU) in vivo. Suppression efficiency of the YFFS tRNAs increases nonlinearly with the amount of injected tRNA, suggesting a significant competition with endogenous, triplet-recognizing tRNA. Both frameshift suppressor tRNAs are less efficient than the amber suppressor tRNA THG73 (Tetrahymena thermophila G73), which has been used extensively for UAA incorporation in Xenopus oocytes. However, the two YFFS tRNAs are more "orthogonal" to the Xenopus system than THG73, and they offer a viable replacement when suppressing at promiscuous sites. To illustrate the potential of combining nonsense and frameshift suppression, we have site-specifically incorporated two and three UAAs simultaneously into a neuroreceptor expressed in vivo.
Anderson,
Exploring the limits of codon and anticodon size.
2002, Pubmed
Anderson,
Exploring the limits of codon and anticodon size.
2002,
Pubmed
Anderson,
An expanded genetic code with a functional quadruplet codon.
2004,
Pubmed
Beene,
Unnatural amino acid mutagenesis in mapping ion channel function.
2003,
Pubmed
,
Xenbase
Dougherty,
Unnatural amino acids as probes of protein structure and function.
2000,
Pubmed
England,
Unnatural amino acid mutagenesis: a precise tool for probing protein structure and function.
2004,
Pubmed
Grosjean,
Guanosine modifications in runoff transcripts of synthetic transfer RNA-Phe genes microinjected into Xenopus oocytes.
1990,
Pubmed
,
Xenbase
Hino,
Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid.
2005,
Pubmed
Hohsaka,
Incorporation of nonnatural amino acids into proteins by using various four-base codons in an Escherichia coli in vitro translation system.
2001,
Pubmed
Kearney,
Determinants of nicotinic receptor gating in natural and unnatural side chain structures at the M2 9' position.
1996,
Pubmed
,
Xenbase
Kearney,
Dose-response relations for unnatural amino acids at the agonist binding site of the nicotinic acetylcholine receptor: tests with novel side chains and with several agonists.
1996,
Pubmed
,
Xenbase
Labarca,
Channel gating governed symmetrically by conserved leucine residues in the M2 domain of nicotinic receptors.
1995,
Pubmed
,
Xenbase
Magliery,
Expanding the genetic code: selection of efficient suppressors of four-base codons and identification of "shifty" four-base codons with a library approach in Escherichia coli.
2001,
Pubmed
Monahan,
Site-specific incorporation of unnatural amino acids into receptors expressed in Mammalian cells.
2003,
Pubmed
Murakami,
Using a solid-phase ribozyme aminoacylation system to reprogram the genetic code.
2003,
Pubmed
Nakamura,
Codon usage tabulated from international DNA sequence databases: status for the year 2000.
2000,
Pubmed
Nowak,
Nicotinic receptor binding site probed with unnatural amino acid incorporation in intact cells.
1995,
Pubmed
,
Xenbase
Nowak,
In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system.
1998,
Pubmed
,
Xenbase
Petersson,
MALDI-TOF mass spectrometry methods for evaluation of in vitro aminoacyl tRNA production.
2002,
Pubmed
Sakamoto,
Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells.
2002,
Pubmed
Saks,
The transfer RNA identity problem: a search for rules.
1994,
Pubmed
Saks,
An engineered Tetrahymena tRNAGln for in vivo incorporation of unnatural amino acids into proteins by nonsense suppression.
1996,
Pubmed
,
Xenbase
Shafer,
Site-specific insertion of spin-labeled L-amino acids in Xenopus oocytes.
2004,
Pubmed
,
Xenbase
Taira,
Four-base codon-mediated incorporation of non-natural amino acids into proteins in a eukaryotic cell-free translation system.
2005,
Pubmed
Zhang,
Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells.
2004,
Pubmed
Zhong,
From ab initio quantum mechanics to molecular neurobiology: a cation-pi binding site in the nicotinic receptor.
1998,
Pubmed
