Orthogonality and Codon Preference of the Pyrrolysyl-tRNA Synthetase-tRNAPyl pair in Escherichia coli for the Genetic Code Expansion
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Systematic studies of basal nonsense suppression, orthogonality of tRNAPyl variants, and cross recognition between codons and tRNA anticodons are reported. E. coli displays detectable basal amber and opal suppression but shows a negligible ochre suppression. Although detectable, basal amber suppression is fully inhibited when a pyrrolysyl-tRNA synthetase (PylRS)-tRNAPyl_CUA pair is genetically encoded. trnaPyl_CUA is aminoacylated by an E. coli aminoacyl-tRNA synthetase at a low level, however, this misaminoacylation is fully inhibited when both PylRS and its substrate are present. Besides that it is fully orthogonal in E. coli and can be coupled with PylRS to genetically incorporate a NAA at an ochre codon, tRNAPyl_UUA is not able to recognize an UAG codon to induce amber suppression. This observation is in direct conflict with the wobble base pair hypothesis and enables using an evolved M. jannaschii tyrosyl-tRNA synthetase-tRNAPyl_UUA pair and the wild type or evolved PylRS-tRNAPyl_UUA pair to genetically incorporate two different NAAs at amber and ochre codons. tRNAPyl_UCA is charged by E. coli tryptophanyl-tRNA synthetase, thus not orthogonal in E. coli. Mutagenic studies of trnaPyl_UCA led to the discovery of its G73U form which shows a higher orthogonality. Mutating trnaPyl_CUA to trnaPyl_UCCU not only leads to the loss of the relative orthogonality of tRNAPyl in E. coli but also abolishes its aminoacylation by PylRS.
PylRS, pyrrolysyl-tRNA synthetase
NAA, noncanonical amino acid
aaRS, aminoacyl-tRNA synthetase
MjTyrRS, Methanococcus jannaschii tyrosyl-tRNA synthetase
T4 PNK, T4 polynucleotide kinase
ESI-MS, electrospray ionization mass spectrometry
PCR, polymerase chain reaction
sfGFP, superfolder green fluorescent protein
sfGFP134TAG, the sfGFP gene with an amber mutation at N134 and a 6×His tag at its C-terminus
sfGFP134TGA,the sfGFP gene with an opal mutation at N134 and a 6×His tag at its C-terminus
sfGFP134TAA,the sfGFP gene with an ochre mutation at N134 and a 6×His tag at its C-terminus
sfGFP134AGGA,the sfGFP gene with an quadruple AGGA mutation at N134 and a 6×His tag at its C-terminus
sfGFP2TAG134TAA, the sfGFP gene with an amber mutation at S2, an ochre mutation at N134, and a 6×His tag at its C-terminus
sfGFP2TGA, the sfGFP gene with an opal mutation at S2 and a 6×His tag at its C-terminus
IPTG, isopropyl -D-thiogalactopyranoside
RF, release factor
TrpRS, tryptophanyl-tRNA synthetase
ArgRS, arginyl-tRNA synthetase
Odoi, Keturah (2012). Orthogonality and Codon Preference of the Pyrrolysyl-tRNA Synthetase-tRNAPyl pair in Escherichia coli for the Genetic Code Expansion. Master's thesis, Texas A&M University. Available electronically from
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