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Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: Minimizing misacylation

Rodriguez, Erik A. and Lester, Henry A. and Dougherty, Dennis A. (2007) Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: Minimizing misacylation. RNA, 13 (10). pp. 1703-1714. ISSN 1355-8382. PMCID PMC1986802. doi:10.1261/rna.666807. https://resolver.caltech.edu/CaltechAUTHORS:20200415-144146916

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1261/rna.666807DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1986802PubMed CentralArticle
ORCID:
AuthorORCID
Lester, Henry A.0000-0002-5470-5255
Dougherty, Dennis A.0000-0003-1464-2461
Additional Information:© 2007 RNA Society. Published by Cold Spring Harbor Laboratory Press. Received May 30, 2007. Accepted July 9, 2007. Published in Advance August 13, 2007. E.A.R. is a National Science Foundation Predoctoral Fellow. This work was supported by the NIH (NS 34407 and NS 11756).
Funders:
Funding AgencyGrant Number
NSF Graduate Research FellowshipUNSPECIFIED
NIHNS 34407
NIHNS 11756
Subject Keywords:nonsense suppression; frameshift suppression; tRNA; opal suppressor tRNA; unnatural amino acid incorporation
Issue or Number:10
PubMed Central ID:PMC1986802
DOI:10.1261/rna.666807
Record Number:CaltechAUTHORS:20200415-144146916
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200415-144146916
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:102569
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:15 Apr 2020 21:50
Last Modified:16 Nov 2021 18:13

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