Amber Suppression in Mammalian Cells Dependent upon Expression of an <i>Escherichia coli</i> Aminoacyl-tRNA Synthetase Gene

Drabkin, Harold J.; Park, Ho‐Jin; RajBhandary, Uttam L.

doi:10.1128/mcb.16.3.907

Cited by 62 publications

(68 citation statements)

References 54 publications

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“…These results are in good agreement with previously published comparisons between bacterial and eukaryotic tyrosyl-tRNA synthetases (17)(18)(19)(20)(21). Similar species specificity has also been observed for several other aminoacyl-tRNA synthetases (55)(56)(57)(58)(59) and is consistent with the hypothesis that the recognition of tRNA by aminoacyl-tRNA synthetases was still evolving after the divergence of bacteria and eukaryotes (39). The observation that tyrosyl-tRNA synthetase from the archaeon M. jannaschii lacks a substantial portion of the tRNA anticodon recognition domain suggests that the archaea may recognize and bind tRNA Tyr in a manner distinct from that found in both bacteria and eukaryotes.…”

Section: Resultssupporting

confidence: 82%

Human Tyrosyl-tRNA Synthetase Shares Amino Acid Sequence Homology with a Putative Cytokine

Kleeman¹,

Wei²,

Simpson

et al. 1997

Journal of Biological Chemistry

116

102

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To test the hypothesis that tRNA Tyr recognition differs between bacterial and human tyrosyl-tRNA synthetases, we sequenced several clones identified as human tyrosyl-tRNA synthetase cDNAs by the Human Genome Project. We found that human tyrosyl-tRNA synthetase is composed of three domains: 1) an aminoterminal Rossmann fold domain that is responsible for formation of the activated E⅐Tyr-AMP intermediate and is conserved among bacteria, archeae, and eukaryotes; 2) a tRNA anticodon recognition domain that has not been conserved between bacteria and eukaryotes; and 3) a carboxyl-terminal domain that is unique to the human tyrosyl-tRNA synthetase and whose primary structure is 49% identical to the putative human cytokine endothelial monocyte-activating protein II, 50% identical to the carboxyl-terminal domain of methionyl-tRNA synthetase from Caenorhabditis elegans, and 43% identical to the carboxyl-terminal domain of Arc1p from Saccharomyces cerevisiae. The first two domains of the human tyrosyl-tRNA synthetase are 52, 36, and 16% identical to tyrosyl-tRNA synthetases from S. cerevisiae, Methanococcus jannaschii, and Bacillus stearothermophilus, respectively. Nine of fifteen amino acids known to be involved in the formation of the tyrosyl-adenylate complex in B. stearothermophilus are conserved across all of the organisms, whereas amino acids involved in the recognition of tRNA Tyr are not conserved. Kinetic analyses of recombinant human and B. stearothermophilus tyrosyl-tRNA synthetases expressed in Escherichia coli indicate that human tyrosyl-tRNA synthetase aminoacylates human but not B. stearothermophilus tRNA Tyr , and vice versa, supporting the original hypothesis. It is proposed that like endothelial monocyte-activating protein II and the carboxyl-terminal domain of Arc1p, the carboxyl-terminal domain of human tyrosyltRNA synthetase evolved from gene duplication of the carboxyl-terminal domain of methionyl-tRNA synthetase and may direct tRNA to the active site of the enzyme.Aminoacyl-tRNA synthetases catalyze the aminoacylation of tRNA by their cognate amino acid. For most aminoacyl-tRNA synthetases (E), tRNA aminoacylation can be separated into two steps: formation of a stable enzyme-bound aminoacyladenylate intermediate (E⅐AA-AMP, Equation 1), followed by transfer of the amino acid (AA) from the aminoacyl-adenylate intermediate to the 3Ј end of the tRNA substrate (Equation 2).The 20 different aminoacyl-tRNA synthetases fall into two distinct structural classes (1, 2). Class I aminoacyl-tRNA synthetases (of which tyrosyl-tRNA synthetase is a member) are characterized by a structurally well conserved amino-terminal Rossmann fold domain which contains the signature sequences "HIGH" and "KMSKS" (3, 4). In contrast, the carboxyl-terminal domains of class I aminoacyl-tRNA synthetases are structurally diverse suggesting that the primordial aminoacyl-tRNA synthetase consisted solely of the amino-terminal Rossmann fold (5-8). Both x-ray crystallography and site-directed mutagenesis of class I aminoacyl-tRNA synthet...

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Section: Resultssupporting

confidence: 82%

Human Tyrosyl-tRNA Synthetase Shares Amino Acid Sequence Homology with a Putative Cytokine

Kleeman¹,

Wei²,

Simpson

et al. 1997

Journal of Biological Chemistry

116

102

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“…Besides providing the only example of a 21st synthetasetRNA pair for use in a eukaryotic system, our approach differs somewhat from that of Schultz and coworkers, in which both the aaRS and the suppressor tRNA are imported from a heterologous organism (13,14), and it offers certain advantages. For example, because the suppressor tRNAs used are derived from tRNA of the same organism or a related organism, transcripts derived from the suppressor tRNA genes are likely to be processed well to yield functional suppressor tRNAs (34). Moreover, the suppressor tRNAs are likely to carry base modifications that are normally found in the tRNA and are, therefore, more likely to be optimally active in suppression (35).…”

Section: Discussionmentioning

confidence: 99%

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

Kowal

Köhrer

RajBhandary

2001

Proc. Natl. Acad. Sci. U.S.A.

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Two critical requirements for developing methods for the sitespecific 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 aaRSsuppressor 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 tyrosyltRNA 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 errorprone 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.

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“…Toward alloprotein synthesis in mammalian cells, nonsense suppression systems have been constructed by the coexpression of a prokaryotic pair of glutaminyltRNA synthetase (GlnRS)⅐tRNA Gln in the cell (26) or by importing the aminoacylated amber suppressor E. coli tRNA Tyr into the cell (45). By using these techniques, the variety of translation systems, together with aaRS engineering, must be further expanded to include mammalian cells.…”

Section: Discussionmentioning

confidence: 99%

“…E. coli glutaminyl-tRNA synthetase and its specific suppressor tRNA have been reported to be orthogonal to the eukaryotic systems (25,26). For TyrRS and tRNA Tyr , the yeast aaRSs hardly aminoacylate the E. coli amber suppressor tRNA Tyr (27,28), whereas the yeast tRNAs, including tRNA Tyr , are not aminoacylated significantly by E. coli TyrRS (29).…”

mentioning

confidence: 99%

An engineered Escherichia coli tyrosyl–tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system

Kiga

Sakamoto

Kodama

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

161

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Tyrosyl-tRNA synthetase (TyrRS) from Escherichia coli was engineered to preferentially recognize 3-iodo-L-tyrosine rather than Ltyrosine for the site-specific incorporation of 3-iodo-L-tyrosine into proteins in eukaryotic translation systems. The wild-type TyrRS does not recognize 3-iodo-L-tyrosine, because of the bulky iodine substitution. On the basis of the reported crystal structure of Bacillus stearothermophilus TyrRS, three residues, Y37, Q179, and Q195, in the L-tyrosine-binding site were chosen for mutagenesis. Thirty-four single amino acid replacements and 16 of their combinations were screened by in vitro biochemical assays. A combination of the Y37V and Q195C mutations changed the amino acid specificity in such a way that the variant TyrRS activates 3-iodo-L-tyrosine 10-fold more efficiently than L-tyrosine. This engineered enzyme, TyrRS(V37C195), was tested for use in the wheat germ cell-free translation system, which has recently been significantly improved, and is now as productive as conventional recombinant systems. During the translation in the wheat germ system, an E. coli suppressor tRNA Tyr was not aminoacylated by the wheat germ enzymes, but was aminoacylated by the E. coli TyrRS(V37C195) variant with 3-iodo-L-tyrosine. After the use of the 3-iodotyrosyl-tRNA in translation, the resultant uncharged tRNA could be aminoacylated again in the system. A mass spectrometric analysis of the produced protein revealed that more than 95% of the amino acids incorporated for an amber codon were iodotyrosine, whose concentration was only twice that of L-tyrosine in the translation. Therefore, the variant enzyme, 3-iodo-L-tyrosine, and the suppressor tRNA can serve as an additional set orthogonal to the 20 endogenous sets in eukaryotic in vitro translation systems.

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Amber Suppression in Mammalian Cells Dependent upon Expression of an Escherichia coli Aminoacyl-tRNA Synthetase Gene

Cited by 62 publications

References 54 publications

Human Tyrosyl-tRNA Synthetase Shares Amino Acid Sequence Homology with a Putative Cytokine

Human Tyrosyl-tRNA Synthetase Shares Amino Acid Sequence Homology with a Putative Cytokine

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

An engineered Escherichia coli tyrosyl–tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system

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