Abstract
Systematic studies of nonsense and sense suppression of the original and three derivative Methanosarcina mazei PylRS-tRNAPyl pairs and cross recognition between nonsense codons and various tRNAPyl anticodons in the Escherichia coli BL21(DE3) cell strain are reported. is orthogonal in E. coli and able to induce strong amber suppression when it is co-expressed with pyrrolysyl-tRNA synthetase (PylRS) and charged with a PylRS substrate, Nε-tert-butoxycarbonyl-l-lysine (BocK). Similar to, is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. Although is expected to recognize a UAG codon based on the wobble hypothesis, the PylRS- pair does not give rise to amber suppression that surpasses the basal amber suppression level in E. coli. E. coli itself displays a relatively high opal suppression level and tryptophan (Trp) is incorporated at an opal mutation site. Although the PylRS- pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. fails to deliver BocK at an AGG codon when co-expressed with PylRS in E. coli.
Highlights
Pyrrolysine (Pyl, Figure 1), the 22nd proteinogenic amino acid that was originally discovered in methanogenic methylamine methyltransferase, is genetically encoded by the RNA nucleotide triplet UAG, a stop codon that halts translation of mRNA during a regular protein translation process [1]
The delivery of Pyl to ribosome is mediated by a unique tRNA, tRNAPCyUlAthat is aminoacylated by a unique aminoacyl-tRNA synthetase, pyrrolysyl-tRNA synthetase (PylRS) [2]. tRNAPCyUlAcontains a special nucleotide triplet CUA, an anticodon that recognizes a UAG stop codon in mRNA
The genetic incorporation of two different noncanonical amino acids (NAAs) into one protein can be potentially applied to install a FRET pair in a protein for conformation and dynamic studies as we demonstrated in a separate publication [26], synthesize proteins with two different posttranslational modifications for their functional investigation, and build phage-displayed peptide libraries with the expanded chemical diversities
Summary
Pyrrolysine (Pyl, Figure 1), the 22nd proteinogenic amino acid that was originally discovered in methanogenic methylamine methyltransferase, is genetically encoded by the RNA nucleotide triplet UAG, a stop codon that halts translation of mRNA during a regular protein translation process [1]. Unlike tRNASec that needs a special elongation factor (SelB in E. coli and EFsec in mammalian cells) and an mRNA secondary structure for its binding to the ribosome A site and recognition of a UGA stop codon for the delivery of selenocysteine, tRNAPCyUlA hijacks the regular translation elongation process to suppress a UAG codon for the incorporation of Pyl [3,4,5]. Previous studies demonstrated that PylRS shows remarkably high substrate promiscuity and is able to charge tRNAPCyUlA with a variety of noncanonical amino acids (NAAs) For these reasons and due to the naturally high orthogonality of the PylRS-tRNAPCyUlA pair in bacteria, yeast, and mammalian cells, this pair has been directly transferred to E. coli, Saccharomyces cerevisiae, and human cells for the genetic incorporation of more than ten lysine derivatives including Ne-tert-butoxycarbonyl-L-lysine (BocK) into proteins at amber mutation sites[6,7,8,9,10,11,12]. The genetic incorporation of two different NAAs into one protein can be potentially applied to install a FRET pair in a protein for conformation and dynamic studies as we demonstrated in a separate publication [26], synthesize proteins with two different posttranslational modifications for their functional investigation, and build phage-displayed peptide libraries with the expanded chemical diversities
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