Nonsense and Sense Suppression Abilities of Original and Derivative Methanosarcina mazei Pyrrolysyl-tRNA Synthetase-tRNAPyl Pairs in the Escherichia coli BL21(DE3) Cell Strain

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.

Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion tool

“…The PylRS-tRNA Pyl pair has also shown high codon flexibility as several alternative codons for coding NCAAs have been demonstrated. [11, 61, 62] This remarkable success is largely attributed to the three unconventional features of PylRS.…”

“…By simply mutating the tRNA Pyl anticodon, we and others have demonstrated that the system can be well applied to reassign other codons such as ochre UAA, opal UGA, and four-base AGGA codons. [61, 62, 127] The PylRS-tRNA Pyl pair also displays remarkable orthogonality toward two engineered tyrosyl-tRNA synthase-tRNA Pyl pairs that were originally from M. jannaschii and E. coli and used to expand the genetic code of bacteria and eukaryotes. By coupling a ${\text{PylRS}-\text{tRNA}}_{\text{UUA}}^{\text{Pyl}}$ pair with these two pairs, two distinctive NCAAs have been genetically encoded at two separate codons namely amber UAG and ochre UAA codons in E. coli and mammalian cells.…”

Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids

“…In this organism, UAG was changed from a stop to a sense codon, provided the appropriate translation machinery was present 21,24 . Nevertheless, a second challenge to multi-site nsAA incorporation by codon reassignment is that the evolved aaRSs show ~100- to 1,000-fold reduced activity 5,19 compared with native enzymes, resulting in inefficient nsAA acylation 19,25,26 , low levels of nsAA-tRNA and low protein yields 21,27,28 , particularly with multi-site nsAA incorporation 20 . We hypothesize that current approaches rely on multi-copy plasmids for aaRS and tRNA overexpression to overcome enzyme inefficiency, which masks differences between modestly and highly active aaRSs capable of multi-site nsAA incorporation.…”

Towards Reassigning the Rare AGG Codon in Escherichia coli

“…Previously we reported that the PylRS-tRNA Pyl pair could be reprogrammed for reassigning three nonsense codons. 5 Here, we wish to show that the same pair can be engineered to reassign the rare AGG codon almost quantitatively to code ncAAs, resolving the low ncAA incorporation efficiency issue that is typically observed in codon reassignment experiments.…”

“…6 By simply mutating the anticodon of tRNA Pyl , we showed that the pair could be successfully reprogrammed to reassign opal and ochre codons for coding ncAAs such as N ε -(t-butyloxycarbonyl)-lysine (BocK), N ε -(allyloxycarbonyl)-lysine (AllocK), and N ε -(t-propargyloxycarbonyl)-lysine (ProK) (Scheme 1). 5 However, when we attempted to use the same strategy to reassign a four-base AGGA codon at the 134 th amino acid site of a sequence-optimized superfolder green fluorescent protein (sfGFP) gene, a full-length protein with Arg134 was predominantly produced, indicating strong frameshift suppression from endogenous arginyl-tRNA Arg . The presence of 5 mM BocK in the growth medium did not lead to a significant level of BocK incorporation at the AGGA codon site.…”