Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 101, Issue 24, Pages 8882-8887Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0307029101
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- NIGMS NIH HHS [GM66494, GM62159, R01 GM062159, F32 GM066494] Funding Source: Medline
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An orthogonal tryptophanyl-transfer RNA (tRNA) synthetase (TrpRS)-mutant opal suppressor tRNA(Trp) (mutRNA(UCA)(Trp)) pair was generated for use in mammalian cells. The anticodon loop of the Bacillus subtilis tRNATrp was mutated to UCA, three positions in the D arm were mutated to generate an internal promoter sequence, and the mutRNA(UCA)(Trp) gene was inserted between the 5' and 3' flanking sequences of the tRNA(Trp-1) gene from Arabidopsis to enhance its expression in mammalian cells. In vitro aminoacylation assays and in vivo opal suppression assays showed that B. subtilis TrpRS (BsTrpRS) charges only the cognate mutRNA(UCA)(Trp) and no endogenous mammalian tRNAs. Similarly, the mutRNA(UCA)(Trp) is specifically charged by B. subtilis TrpRS and not by endogenous synthetases in mammalian cells. Site-directed mutagenesis was then used to alter the specificity of BsTrpRS to uniquely charge 5-hydoxy-L-tryptophan. The resulting mutant BsTrpRS-mutRNA(UCA)(Trp) pair allows the efficient and selective incorporation of 5-hydroxy-L-tryptophan into mammalian proteins in response to the codon, TGA. This amino acid can be used as a fluorescence probe and also undergoes electrochemical oxidation in situ to generate an efficient protein crosslinking.
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