RS/tRNA Foundational Publication Support
Kiga, Daisuke, Kensaku Sakamoto, Koichiro Kodama, Takanori Kigawa, Takayoshi Matsuda, Takashi Yabuki, Mikako Shirouzu, et al. (2002) 2002. “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”. Proceedings Of The National Academy Of Sciences Of The United States Of America. 99: 5. doi:10.1073/pnas.142220099.
Sakamoto, Kensaku, Akiko Hayashi, Ayako Sakamoto, Daisuke Kiga, Hiroshi Nakayama, Akiko Soma, Takatsugu Kobayashi, et al. (2002) 2002. “Site-Specific Incorporation Of An Unnatural Amino Acid Into Proteins In Mammalian Cells.”. Nucleic Acids Research 30 (21): 4692-9.
Iraha, Fumie, Kenji Oki, Takatsugu Kobayashi, Satoshi Ohno, Takashi Yokogawa, Kazuya Nishikawa, Shigeyuki Yokoyama, and Kensaku Sakamoto. (2010) 2010. “Functional Replacement Of The Endogenous Tyrosyl-Trna Synthetase-Trnatyr Pair By The Archaeal Tyrosine Pair In Escherichia Coli For Genetic Code Expansion.”. Nucleic Acids Research 38 (11): 3682-91. doi:10.1093/nar/gkq080.
RS/tRNA Protocols and Structural Information
Hino, Nobumasa, Akiko Hayashi, Kensaku Sakamoto, and Shigeyuki Yokoyama. (2006) 2006. “Site-Specific Incorporation Of Non-Natural Amino Acids Into Proteins In Mammalian Cells With An Expanded Genetic Code.”. Nature Protocols 1 (6): 2957-62.
Iraha, Fumie, Kenji Oki, Takatsugu Kobayashi, Satoshi Ohno, Takashi Yokogawa, Kazuya Nishikawa, Shigeyuki Yokoyama, and Kensaku Sakamoto. (2010) 2010. “Functional Replacement Of The Endogenous Tyrosyl-Trna Synthetase-Trnatyr Pair By The Archaeal Tyrosine Pair In Escherichia Coli For Genetic Code Expansion.”. Nucleic Acids Research 38 (11): 3682-91. doi:10.1093/nar/gkq080.
RS/tRNA Pair Development Year
2002
ncAA(s) Incorporated
3-iodo-L-tyrosine
ncAA Structure (png, jpg, jpeg)
ncAA Utility
for anomalous dispersion phasing in protein crystallography.
3-Azido-L-tyrosine
ncAA Structure (png, jpg, jpeg)
ncAA Utility
n/a
RS Organism of Origin
Parent RS
RS Mutations
Y37V
Q195C
Q195C
tRNA Organism of Origin
Parent tRNA
tRNA Anticodon
CUA
Multiple tRNAs?
In second 2002 foundational paper, found that for expression in CHO and HEK293 cells the Tyr tRNA did not work, and a B stearothermophilius Tyr tRNA did work.
RS/tRNA Availability
N/A
Used in what cell line?
RS/tRNA Additional Notes
First 2002 paper used the RS in a wheat germ cell free translation system, and the second 2002 foundational paper used it in mammalian cells using 0.3 mM 3-I-tyr to yield high-fidelity incorporation into ras(32) at ~20% efficiency. Also created a CHO-Y stable cell line with the RS.
The 2010 paper showed it could also work in an E coli "FB3" strain with the genomic TyrRS/tRNA genes removed and archael TyrRS/tRNA genes supplied (tyrU tyrV ΔtyrS ΔtyrT pMJY]. The RS incorporated both 3-iodo and 3-azido-Tyr well and with high fidelity and by gel appearing to be ~50 and ~100% efficiency respectively. A crystal structure of the RS-3AzTyr complex is also reported (PDB code 2YXN).
The 2010 paper showed it could also work in an E coli "FB3" strain with the genomic TyrRS/tRNA genes removed and archael TyrRS/tRNA genes supplied (tyrU tyrV ΔtyrS ΔtyrT pMJY]. The RS incorporated both 3-iodo and 3-azido-Tyr well and with high fidelity and by gel appearing to be ~50 and ~100% efficiency respectively. A crystal structure of the RS-3AzTyr complex is also reported (PDB code 2YXN).