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.
RNA
2007 Oct 01;1310:1703-14. doi: 10.1261/rna.666807.
Show Gene links
Show Anatomy links
Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: minimizing misacylation.
Rodriguez EA, Lester HA, Dougherty DA.
???displayArticle.abstract???
The incorporation of unnatural amino acids site-specifically is a valuable technique for structure-function studies, incorporation of biophysical probes, and determining protein-protein interactions. THG73 is an amber suppressor tRNA used extensively for the incorporation of >100 different residues in over 20 proteins, but under certain conditions THG73 is aminoacylated in vivo by endogenous aminoacyl-tRNA synthetase. Similar aminoacylation is seen with the Escherichia coli Asn amber suppressor tRNA, which has also been used to incorporate UAAs in many studies. We now find that the natural amino acid placed on THG73 is Gln. Since the E. coli GlnRS recognizes positions in the acceptor stem, we made several acceptor stem mutations in the second to fourth positions on THG73. All mutations reduce aminoacylation in vivo and allow for the selection of highly orthogonal tRNAs. To show the generality of these mutations, we created opal suppressor tRNAs that show less aminoacylation in Xenopus oocytes relative to THG73. We have created a library of Tetrahymena thermophila Gln amber suppressor tRNAs that will be useful for determining optimal suppressor tRNAs for use in other eukaryotic cells.
Anderson,
Adaptation of an orthogonal archaeal leucyl-tRNA and synthetase pair for four-base, amber, and opal suppression.
2003, Pubmed
Anderson,
Adaptation of an orthogonal archaeal leucyl-tRNA and synthetase pair for four-base, amber, and opal suppression.
2003,
Pubmed Beene,
Unnatural amino acid mutagenesis in mapping ion channel function.
2003,
Pubmed
,
Xenbase Brejc,
Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors.
2001,
Pubmed Capone,
Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian cells.
1986,
Pubmed Cload,
Development of improved tRNAs for in vitro biosynthesis of proteins containing unnatural amino acids.
1996,
Pubmed Dougherty,
Unnatural amino acids as probes of protein structure and function.
2000,
Pubmed England,
Mapping disulfide connectivity using backbone ester hydrolysis.
1999,
Pubmed
,
Xenbase Filatov,
The role of conserved leucines in the M2 domain of the acetylcholine receptor in channel gating.
1995,
Pubmed
,
Xenbase Frugier,
Sequences outside recognition sets are not neutral for tRNA aminoacylation. Evidence for nonpermissive combinations of nucleotides in the acceptor stem of yeast tRNAPhe.
1998,
Pubmed Fukunaga,
A base pair at the bottom of the anticodon stem is reciprocally preferred for discrimination of cognate tRNAs by Escherichia coli lysyl- and glutaminyl-tRNA synthetases.
2006,
Pubmed Gallivan,
Site-specific incorporation of biotinylated amino acids to identify surface-exposed residues in integral membrane proteins.
1997,
Pubmed
,
Xenbase Hanyu,
Dramatic events in ciliate evolution: alteration of UAA and UAG termination codons to glutamine codons due to anticodon mutations in two Tetrahymena tRNAs.
1986,
Pubmed Ibba,
Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme.
1996,
Pubmed Jahn,
Anticodon and acceptor stem nucleotides in tRNA(Gln) are major recognition elements for E. coli glutaminyl-tRNA synthetase.
1991,
Pubmed Kearney,
Determinants of nicotinic receptor gating in natural and unnatural side chain structures at the M2 9' position.
1996,
Pubmed
,
Xenbase Kleina,
Construction of Escherichia coli amber suppressor tRNA genes. II. Synthesis of additional tRNA genes and improvement of suppressor efficiency.
1990,
Pubmed Köhrer,
Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells.
2004,
Pubmed Kosolapov,
Acetylcholine receptor gating is influenced by the polarity of amino acids at position 9' in the M2 domain.
2000,
Pubmed
,
Xenbase Liu,
Characterization of an 'orthogonal' suppressor tRNA derived from E. coli tRNA2(Gln).
1997,
Pubmed McClain,
The importance of tRNA backbone-mediated interactions with synthetase for aminoacylation.
1998,
Pubmed Murakami,
Using a solid-phase ribozyme aminoacylation system to reprogram the genetic code.
2003,
Pubmed Murakami,
A highly flexible tRNA acylation method for non-natural polypeptide synthesis.
2006,
Pubmed Nakata,
Binding efficiency of elongation factor Tu to tRNAs charged with nonnatural fluorescent amino acids.
2006,
Pubmed Normanly,
Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity.
1990,
Pubmed Nowak,
In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system.
1998,
Pubmed
,
Xenbase Perona,
Structural basis for misaminoacylation by mutant E. coli glutaminyl-tRNA synthetase enzymes.
1989,
Pubmed Petersson,
MALDI-TOF mass spectrometry methods for evaluation of in vitro aminoacyl tRNA production.
2002,
Pubmed Robertson,
The use of 5'-phospho-2 deoxyribocytidylylriboadenosine as a facile route to chemical aminoacylation of tRNA.
1989,
Pubmed Rodriguez,
Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 2: evaluating suppression efficiency.
2007,
Pubmed
,
Xenbase Rodriguez,
In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression.
2006,
Pubmed
,
Xenbase Rothman,
Caged phosphoproteins.
2005,
Pubmed Saks,
An engineered Tetrahymena tRNAGln for in vivo incorporation of unnatural amino acids into proteins by nonsense suppression.
1996,
Pubmed
,
Xenbase Sasaki,
Regulation mechanisms of intracellular pH of Xenopus laevis oocyte.
1992,
Pubmed
,
Xenbase Shafer,
Site-specific insertion of spin-labeled L-amino acids in Xenopus oocytes.
2004,
Pubmed
,
Xenbase Unwin,
Refined structure of the nicotinic acetylcholine receptor at 4A resolution.
2005,
Pubmed Wang,
Tandemly activated tRNAs as participants in protein synthesis.
2006,
Pubmed Webb,
Direct measurement of intracellular pH changes in Xenopus eggs at fertilization and cleavage.
1981,
Pubmed
,
Xenbase Whitfield,
Nonsense-mediated mRNA decay in Xenopus oocytes and embryos.
1994,
Pubmed
,
Xenbase 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
