RS/tRNA Foundational Publication Support
Amiram, Miriam, Adrian D. Haimovich, Chenguang Fan, Yane-Shih Wang, Hans-Rudolf Aerni, Ioanna Ntai, Daniel W. Moonan, et al. (dec) 2015. “Evolution Of Translation Machinery In Recoded Bacteria Enables Multi-Site Incorporation Of Nonstandard Amino Acids”. Nature Biotechnology 33: 1272-1279. doi:10.1038/nbt.3372.
RS/tRNA Pair Development Year
2015
ncAA(s) Incorporated
p-acetyl-l-phenylalanine
ncAA Structure (png, jpg, jpeg)
ncAA Utility
Site-directed spin labeling, electron paramagnetic resonance
p-azido-L-phenylalanine (pAzF)
ncAA Structure (png, jpg, jpeg)
ncAA Utility
Used as a photocrosslinker, allowing for crosslinking and bioorthogonal ligation of protein.
p-Iodo-L-phenylalanine
ncAA Structure (png, jpg, jpeg)
ncAA Utility
Mutant GFPexcitation/ emission, later used for protein crystallization and as reactive handle. Structural determination of proteins. Amber suppression in a TB model mycobacterium
Trifluoromethyl-L phenylalanine
ncAA Structure (png, jpg, jpeg)
ncAA Utility
19F Probe for protein
NMR
NMR
O-tert-butyl-L-tyrosine
ncAA Structure (png, jpg, jpeg)
ncAA Utility
n/a
RS Organism of Origin
Parent RS
RS Mutations
L65V
A167D
A167D
tRNA Organism of Origin
Parent tRNA
tRNA Anticodon
CUA
Other tRNA Mutations
R257G
RS/tRNA Availability
Can be purchased from Addgene Plasmid #73544
Used in what cell line?
RS/tRNA Additional Notes
The RS was evolved from the pAcFRS using MAGE (Multiplex automated genome engineering) in WT-E.coli .
It showed a 15-fold increased expression of GFP(3UAG) [ELP(Elastin-Like-Protein(30UAG)-GFP] compared to pAcFRS, the Selectivity testing showed a distinct preference for pAcF over pAzf. However, this was shown to diminish when exposed in a multicopy. The incorporation was shown to be relatively high after analysis of GFP fluorescence of ELP (30UAG)-GFP at 0.25mM, 0.5mM and 1mM of pAcF. This Assay also showed a modest kcat/Km improvement from pAcFRS with a 8.7-fold increase for tRNA aminoacylation.
By examing ELP(30UAG) and ELP(10UAG)-GFP with shotgun liquid chromatography(LC)-tandem MS showed pAcF incorporation >95% for all peptide and producing full length protein >97% of time.
The changes to the binding pocket had allowed to incorporation of the following ncAA at the same level as pAcF: L-4-Methoxy-phenylalanine(MeY) as assayed by GFP(3UAG) fluorescence. While these were incorporated at higher rate: L-4-Iodo-phenylalanine (4IF), O-tert-Butyl_L_tyrosine(BuY) and 4-phenyl-phenylalanine(phF) as assayed by GFP(3UAG) fluorescence.
It showed a 15-fold increased expression of GFP(3UAG) [ELP(Elastin-Like-Protein(30UAG)-GFP] compared to pAcFRS, the Selectivity testing showed a distinct preference for pAcF over pAzf. However, this was shown to diminish when exposed in a multicopy. The incorporation was shown to be relatively high after analysis of GFP fluorescence of ELP (30UAG)-GFP at 0.25mM, 0.5mM and 1mM of pAcF. This Assay also showed a modest kcat/Km improvement from pAcFRS with a 8.7-fold increase for tRNA aminoacylation.
By examing ELP(30UAG) and ELP(10UAG)-GFP with shotgun liquid chromatography(LC)-tandem MS showed pAcF incorporation >95% for all peptide and producing full length protein >97% of time.
The changes to the binding pocket had allowed to incorporation of the following ncAA at the same level as pAcF: L-4-Methoxy-phenylalanine(MeY) as assayed by GFP(3UAG) fluorescence. While these were incorporated at higher rate: L-4-Iodo-phenylalanine (4IF), O-tert-Butyl_L_tyrosine(BuY) and 4-phenyl-phenylalanine(phF) as assayed by GFP(3UAG) fluorescence.