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
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.