RS/tRNA Foundational Publication Support
Gautier, Arnaud, Duy P Nguyen, Hrvoje Lusic, Wenlin An, Alexander Deiters, and Jason W Chin. (2010) 2010. “Genetically Encoded Photocontrol Of Protein Localization In Mammalian Cells.”. Journal Of The American Chemical Society 132 (12): 4086-8. doi:10.1021/ja910688s.
Hancock, S. M., R. Uprety, A. Deiters, and J. W. Chin. n.d. “Expanding The Genetic Code Of Yeast For Incorporation Of Diverse Unnatural Amino Acids Via A Pyrrolysyl-Trna Synthetase/Trna Pair” 132: 14819+.
RS/tRNA Pair Development Year
2010
ncAA(s) Incorporated
N-epsilon-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)-L-lysine
ncAA Structure (png, jpg, jpeg)
![N-epsilon-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)-L-lysine](/sites/gce4all/files/styles/medium/public/2025-01/Page_20_Structure_3.png?itok=19HIL70R)
ncAA Utility
Subst. of NLS for photocaged nuclear localization of GFP and p53. Also utilized for Light-activated gene
expression
expression
RS Organism of Origin
Parent RS
RS Mutations
M241F
A267S
Y271C
L274M
A267S
Y271C
L274M
tRNA Organism of Origin
Parent tRNA
tRNA Anticodon
CUA
Multiple tRNAs?
To encode PCKRS into yeast, the the G3·U70 base pair in MbtRNACUA^Pyl was converted to A3-U70.
RS/tRNA Availability
N/A
RS/tRNA Additional Notes
In mammalian Hek-293 cells, PCKRS successfully incorporated N-epsilon-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)-L-lysine as confirmed by fluorescence confocal micrographs of Hek-293 cells containing EGFP. This result was also confirmed by immunoblotting.
PCKRS allowed cells to survive in 300μg/mL chloramphenicol media in the presence of N-epsilon-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)-L-lysine, but did not survive in its absence, showing high specificity.
Strong applicability in mammalian studies shown by full length protein expression of nls-gfp-ha incorporating N-epsilon-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)-L-lysine dependent upon the presence of PCKRS. Confirmed by fluorescence imaging.
Using the tRNA mutation listed, amino acid incorporation into purified SOD was shown in the presence of N-epsilon-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)-L-lysine and PCKRS. hSOD yields used were 30−100 μg/L of yeast culture, showing the possibility of incorporation into yeast although MS data in yeast had not yet been acquired.
PCKRS allowed cells to survive in 300μg/mL chloramphenicol media in the presence of N-epsilon-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)-L-lysine, but did not survive in its absence, showing high specificity.
Strong applicability in mammalian studies shown by full length protein expression of nls-gfp-ha incorporating N-epsilon-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)-L-lysine dependent upon the presence of PCKRS. Confirmed by fluorescence imaging.
Using the tRNA mutation listed, amino acid incorporation into purified SOD was shown in the presence of N-epsilon-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)-L-lysine and PCKRS. hSOD yields used were 30−100 μg/L of yeast culture, showing the possibility of incorporation into yeast although MS data in yeast had not yet been acquired.