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Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria.
Proc Natl Acad Sci U S A. 2001 Feb 27; 98(5):2268-73.PN

Abstract

Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are (i) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs) and (ii) an aminoacyl-tRNA synthetase that aminoacylates the suppressor tRNA but no other tRNA in the cell. Here we describe two such aaRS-suppressor tRNA pairs, one for use in the yeast Saccharomyces cerevisiae and another for use in Escherichia coli. The "21st synthetase-tRNA pairs" include E. coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyl-tRNA synthetase (TyrRS) along with an amber suppressor derived from E. coli initiator tRNA, for use in E. coli. The suppressor tRNAs are aminoacylated in vivo only in the presence of the heterologous aaRSs, and the aminoacylated tRNAs function efficiently in suppression of amber codons. Plasmids carrying the E. coli GlnRS gene can be stably maintained in yeast. However, plasmids carrying the yeast TyrRS gene could not be stably maintained in E. coli. This lack of stability is most likely due to the fact that the wild-type yeast TyrRS misaminoacylates the E. coli proline tRNA. By using error-prone PCR, we have isolated and characterized three mutants of yeast TyrRS, which can be stably expressed in E. coli. These mutants still aminoacylate the suppressor tRNA essentially quantitatively in vivo but show increased discrimination in vitro for the suppressor tRNA over the E. coli proline tRNA by factors of 2.2- to 6.8-fold.

Authors+Show Affiliations

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

11226228

Citation

Kowal, A K., et al. "Twenty-first aminoacyl-tRNA Synthetase-suppressor tRNA Pairs for Possible Use in Site-specific Incorporation of Amino Acid Analogues Into Proteins in Eukaryotes and in Eubacteria." Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 5, 2001, pp. 2268-73.
Kowal AK, Kohrer C, RajBhandary UL. Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria. Proc Natl Acad Sci USA. 2001;98(5):2268-73.
Kowal, A. K., Kohrer, C., & RajBhandary, U. L. (2001). Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria. Proceedings of the National Academy of Sciences of the United States of America, 98(5), 2268-73.
Kowal AK, Kohrer C, RajBhandary UL. Twenty-first aminoacyl-tRNA Synthetase-suppressor tRNA Pairs for Possible Use in Site-specific Incorporation of Amino Acid Analogues Into Proteins in Eukaryotes and in Eubacteria. Proc Natl Acad Sci USA. 2001 Feb 27;98(5):2268-73. PubMed PMID: 11226228.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria. AU - Kowal,A K, AU - Kohrer,C, AU - RajBhandary,U L, Y1 - 2001/01/23/ PY - 2001/2/28/pubmed PY - 2001/9/14/medline PY - 2001/2/28/entrez SP - 2268 EP - 73 JF - Proceedings of the National Academy of Sciences of the United States of America JO - Proc. Natl. Acad. Sci. U.S.A. VL - 98 IS - 5 N2 - Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are (i) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs) and (ii) an aminoacyl-tRNA synthetase that aminoacylates the suppressor tRNA but no other tRNA in the cell. Here we describe two such aaRS-suppressor tRNA pairs, one for use in the yeast Saccharomyces cerevisiae and another for use in Escherichia coli. The "21st synthetase-tRNA pairs" include E. coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyl-tRNA synthetase (TyrRS) along with an amber suppressor derived from E. coli initiator tRNA, for use in E. coli. The suppressor tRNAs are aminoacylated in vivo only in the presence of the heterologous aaRSs, and the aminoacylated tRNAs function efficiently in suppression of amber codons. Plasmids carrying the E. coli GlnRS gene can be stably maintained in yeast. However, plasmids carrying the yeast TyrRS gene could not be stably maintained in E. coli. This lack of stability is most likely due to the fact that the wild-type yeast TyrRS misaminoacylates the E. coli proline tRNA. By using error-prone PCR, we have isolated and characterized three mutants of yeast TyrRS, which can be stably expressed in E. coli. These mutants still aminoacylate the suppressor tRNA essentially quantitatively in vivo but show increased discrimination in vitro for the suppressor tRNA over the E. coli proline tRNA by factors of 2.2- to 6.8-fold. SN - 0027-8424 UR - https://www.unboundmedicine.com/medline/citation/11226228/Twenty_first_aminoacyl_tRNA_synthetase_suppressor_tRNA_pairs_for_possible_use_in_site_specific_incorporation_of_amino_acid_analogues_into_proteins_in_eukaryotes_and_in_eubacteria_ L2 - http://www.pnas.org/cgi/pmidlookup?view=long&pmid=11226228 DB - PRIME DP - Unbound Medicine ER -