Phosphoserine aminoacylation of tRNA bearing an unnatural base anticodon

Ryuya Fukunaga; Yoko Harada; Ichiro Hirao; Shigeyuki Yokoyama

doi:10.1016/j.bbrc.2008.05.078

Phosphoserine aminoacylation of tRNA bearing an unnatural base anticodon

Ryuya Fukunaga, Yoko Harada, Ichiro Hirao, Shigeyuki Yokoyama

Research output: Contribution to journal › Article › peer-review

Abstract

An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) could expand the genetic alphabet and allow the incorporation of non-standard amino acids into proteins at defined positions. For this purpose, we synthesized tRNAs bearing Pa at the anticodon and tested non-standard amino acid phosphoserine aminoacylation by the wild-type and various engineered phosphoseryl-tRNA synthetases (SepRSs). The D418N D420N T423V triple mutant of SepRS efficiently charged phosphoserine to tRNA containing the PaUA anticodon with a K_m = 47.1 μM and a k_cat = 0.151 s^-1, which are comparable to the values of the wild-type SepRS for its cognate substrate, tRNA^Cys with the GCA anticodon (26.9 μM and 0.111 s^-1). The triple mutant SepRS and the tRNA with the PaUA anticodon represent a specific pair for the site-specific incorporation of phosphoserine into proteins in response to the UADs codon within mRNA.

Original language	English (US)
Pages (from-to)	480-485
Number of pages	6
Journal	Biochemical and Biophysical Research Communications
Volume	372
Issue number	3
DOIs	https://doi.org/10.1016/j.bbrc.2008.05.078
State	Published - Aug 1 2008
Externally published	Yes

Keywords

Aminoacylation
Engineering
Phosphoserine
Synthetase
Unnatural base pair
tRNA

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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10.1016/j.bbrc.2008.05.078

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title = "Phosphoserine aminoacylation of tRNA bearing an unnatural base anticodon",

abstract = "An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) could expand the genetic alphabet and allow the incorporation of non-standard amino acids into proteins at defined positions. For this purpose, we synthesized tRNAs bearing Pa at the anticodon and tested non-standard amino acid phosphoserine aminoacylation by the wild-type and various engineered phosphoseryl-tRNA synthetases (SepRSs). The D418N D420N T423V triple mutant of SepRS efficiently charged phosphoserine to tRNA containing the PaUA anticodon with a Km = 47.1 μM and a kcat = 0.151 s-1, which are comparable to the values of the wild-type SepRS for its cognate substrate, tRNACys with the GCA anticodon (26.9 μM and 0.111 s-1). The triple mutant SepRS and the tRNA with the PaUA anticodon represent a specific pair for the site-specific incorporation of phosphoserine into proteins in response to the UADs codon within mRNA.",

keywords = "Aminoacylation, Engineering, Phosphoserine, Synthetase, Unnatural base pair, tRNA",

author = "Ryuya Fukunaga and Yoko Harada and Ichiro Hirao and Shigeyuki Yokoyama",

note = "Funding Information: This work was supported by Grants-in-Aid for Scientific Research in Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, and the RIKEN Structural Genomics/Proteomics Initiative (RSGI) of the National Project on Protein Structural and Functional Analyses, MEXT. R.F. was supported by Research Fellowships from the Japan Society for the Promotion of Science. We thank Akira Sato and Tsuneo Mitsui for synthesizing the nucleoside derivatives. I.H. was supported by a Grant-in-Aid for Scientific Research (KAKENHI 19201046) from the Ministry of Education, Culture, Sports, Science, and Technology.",

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doi = "10.1016/j.bbrc.2008.05.078",

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AU - Fukunaga, Ryuya

AU - Harada, Yoko

AU - Hirao, Ichiro

AU - Yokoyama, Shigeyuki

N1 - Funding Information: This work was supported by Grants-in-Aid for Scientific Research in Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, and the RIKEN Structural Genomics/Proteomics Initiative (RSGI) of the National Project on Protein Structural and Functional Analyses, MEXT. R.F. was supported by Research Fellowships from the Japan Society for the Promotion of Science. We thank Akira Sato and Tsuneo Mitsui for synthesizing the nucleoside derivatives. I.H. was supported by a Grant-in-Aid for Scientific Research (KAKENHI 19201046) from the Ministry of Education, Culture, Sports, Science, and Technology.

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N2 - An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) could expand the genetic alphabet and allow the incorporation of non-standard amino acids into proteins at defined positions. For this purpose, we synthesized tRNAs bearing Pa at the anticodon and tested non-standard amino acid phosphoserine aminoacylation by the wild-type and various engineered phosphoseryl-tRNA synthetases (SepRSs). The D418N D420N T423V triple mutant of SepRS efficiently charged phosphoserine to tRNA containing the PaUA anticodon with a Km = 47.1 μM and a kcat = 0.151 s-1, which are comparable to the values of the wild-type SepRS for its cognate substrate, tRNACys with the GCA anticodon (26.9 μM and 0.111 s-1). The triple mutant SepRS and the tRNA with the PaUA anticodon represent a specific pair for the site-specific incorporation of phosphoserine into proteins in response to the UADs codon within mRNA.

AB - An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) could expand the genetic alphabet and allow the incorporation of non-standard amino acids into proteins at defined positions. For this purpose, we synthesized tRNAs bearing Pa at the anticodon and tested non-standard amino acid phosphoserine aminoacylation by the wild-type and various engineered phosphoseryl-tRNA synthetases (SepRSs). The D418N D420N T423V triple mutant of SepRS efficiently charged phosphoserine to tRNA containing the PaUA anticodon with a Km = 47.1 μM and a kcat = 0.151 s-1, which are comparable to the values of the wild-type SepRS for its cognate substrate, tRNACys with the GCA anticodon (26.9 μM and 0.111 s-1). The triple mutant SepRS and the tRNA with the PaUA anticodon represent a specific pair for the site-specific incorporation of phosphoserine into proteins in response to the UADs codon within mRNA.

KW - Aminoacylation

KW - Engineering

KW - Phosphoserine

KW - Synthetase

KW - Unnatural base pair

KW - tRNA

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ER -

Phosphoserine aminoacylation of tRNA bearing an unnatural base anticodon

Abstract

Keywords

ASJC Scopus subject areas

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