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Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 2: Evaluating suppression efficiency

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 2: Evaluating suppression efficiency. RNA, 13 (10). pp. 1715-1722. ISSN 1355-8382. PMCID PMC1986817. doi:10.1261/rna.667607. https://resolver.caltech.edu/CaltechAUTHORS:20200415-144744313

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Abstract

The incorporation of unnatural amino acids into proteins is a valuable tool for addition of biophysical probes, bio-orthogonal functionalities, and photoreactive cross-linking agents, although these approaches often require quantities of protein that are difficult to access with chemically aminoacylated tRNAs. THG73 is an amber suppressor tRNA that has been used extensively, incorporating over 100 residues in 20 proteins. In vitro studies have shown that the Escherichia coli Asn amber suppressor (ENAS) suppresses better than THG73. However, we report here that ENAS suppresses with <26% of the efficiency of THG73 in Xenopus oocytes. We then tested the newly developed Tetrahymena thermophila Gln amber suppressor (TQAS) tRNA library, which contains mutations in the second to fourth positions of the acceptor stem. The acceptor stem mutations have no adverse effect on suppression efficiency and, in fact, can increase the suppression efficiency. Combining mutations causes an averaging of suppression efficiency, and increased suppression efficiency does not correlate with increased ΔG of the acceptor stem. We created a T. thermophila opal suppressor, TQOpS′, which shows ∼50% suppression efficiency relative to THG73. The TQAS tRNA library, composed of functional suppressor tRNAs, has been created and will allow for screening in eukaryotic cells, where rapid analysis of large libraries is not feasible.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1261/rna.667607DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1986817PubMed 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:PMC1986817
DOI:10.1261/rna.667607
Record Number:CaltechAUTHORS:20200415-144744313
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200415-144744313
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:102570
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:15 Apr 2020 21:55
Last Modified:16 Nov 2021 18:13

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