RS/tRNA Foundational Publication Support
Tanrikulu, Caglar, Emmanuelle Schmitt, Yves Mechulam, William A Goddard, and David A Tirrell. (2009) 2009. “Discovery Of Escherichia Coli Methionyl-Trna Synthetase Mutants For Efficient Labeling Of Proteins With Azidonorleucine In Vivo.”. Proceedings Of The National Academy Of Sciences Of The United States Of America 106 (36): 15285-90. doi:10.1073/pnas.0905735106.
Grammel, Markus, Mingzi Zhang, and Howard C Hang. (2010) 2010. “Orthogonal Alkynyl Amino Acid Reporter For Selective Labeling Of Bacterial Proteomes During Infection.”. Angewandte Chemie (International Ed. In English) 49 (34): 5970-4. doi:10.1002/anie.201002050.
Ngo, John T, Erin M Schuman, and David A Tirrell. (2013) 2013. “Mutant Methionyl-Trna Synthetase From Bacteria Enables Site-Selective N-Terminal Labeling Of Proteins Expressed In Mammalian Cells.”. Proceedings Of The National Academy Of Sciences Of The United States Of America 110 (13): 4992-7. doi:10.1073/pnas.1216375110.
RS/tRNA Usage Publications
Ngo, John T, Julie Champion, Alborz Mahdavi, Caglar Tanrikulu, Kimberly Beatty, Rebecca Connor, Tae Hyeon Yoo, Daniela C Dieterich, Erin M Schuman, and David A Tirrell. (2009) 2009. “Cell-Selective Metabolic Labeling Of Proteins.”. Nature Chemical Biology 5 (10): 715-7. doi:10.1038/nchembio.200.
Mahdavi, Alborz, Janek Szychowski, John T Ngo, Michael J Sweredoski, Robert L J Graham, Sonja Hess, Olaf Schneewind, Sarkis K Mazmanian, and David A Tirrell. (2014) 2014. “Identification Of Secreted Bacterial Proteins By Noncanonical Amino Acid Tagging.”. Proceedings Of The National Academy Of Sciences Of The United States Of America 111 (1): 433-8. doi:10.1073/pnas.1301740111.
RS/tRNA Pair Development Year
2009
ncAA(s) Incorporated
Azidonorleucine
ncAA Structure (png, jpg, jpeg)
ncAA Utility
Biorthogonal Ligation, Click Chemistry labeling with fluorescence or affinity labels. Specifically used for cell-specific proteomics studies using "bioorthogonal noncanonical amino acid tagging" (BONCAT) proteomics. Contrasts with Azidohomoalanine which does not allow cell-specific studies.
RS Organism of Origin
Parent RS
RS Mutations
L13N
Y306L
H301L
Y306L
H301L
tRNA Organism of Origin
Parent tRNA
tRNA Anticodon
UAC
Multiple tRNAs?
Also works with Salmonella tRNA Met and with the mammalian initiator tRNA Met
RS/tRNA Availability
Addgene Plasmid #51401
Used in what cell line?
RS/tRNA Additional Notes
The RS was selected to work at low ncAA and high Met concentrations to allow for cell-specific proteomics. The PLL mutant performed similarly but was not pursued further. In DH10B E coli incorporated Anl at a 10-20% level in place of Met, enough to do proteomics.
In Y.enterocolitica, used Anl incorporation and ligation with an alkyne-functionalized TAMRA dye to identify secreted proteins both in the absence of host cells and in the presence of Hela host cells.
In HEK cells only incorporates into N-terminal Met sites, and UP50 for incorporation was ~0.5 mM. Authors were not able to get working in C. elegans.
For broad replacement of Met with Anl in eukayotic cells the dMetRS(L262G) and the L274G MmMetRS were developed.
2010 paper shows RS in Salmonella incorporates AOA better than ANL, providing an alternative click chemistry ncAA for proteomics studies.
In Y.enterocolitica, used Anl incorporation and ligation with an alkyne-functionalized TAMRA dye to identify secreted proteins both in the absence of host cells and in the presence of Hela host cells.
In HEK cells only incorporates into N-terminal Met sites, and UP50 for incorporation was ~0.5 mM. Authors were not able to get working in C. elegans.
For broad replacement of Met with Anl in eukayotic cells the dMetRS(L262G) and the L274G MmMetRS were developed.
2010 paper shows RS in Salmonella incorporates AOA better than ANL, providing an alternative click chemistry ncAA for proteomics studies.