Pyrrolysine (Pyl), the 22nd amino acid, is genetically encoded at amber codon through the pyrrolysyl-tRNA synthetase (PylRS)�amber suppressing tRNA (pylTCUA) pair in certain methanogenic archaea and some bacteria. Although the original and evolved PylRS- pylTCUA pairs transferred to E. coli have enabled the genetic incorporation of many non-canonical amino acids (NAAs) into proteins, the system needs to be further investigated to determine whether mutant pylT forms can be directly used to genetically incorporate NAAs at an ochre, opal or four base (AGGA) codon. The suppression of the original and three derivative Methanosarcina mazei PylRS- pylT pairs between the amber, ochre, opal, four base codon and the pylT mutants (CUA, UUA, UCA, and UCCU) were characterized by the incorporation of the PylRS substrate, Ne-tert-butoxycarbonyl-L-lysine (BocK). pylTCUA is orthogonal in E. coli and able to induce strong amber suppression when it is co-expressed with PylRS and charged with BocK. Similar to pylTCUA, pylTUUA is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. While pylTCUA and pylTUUA are both orthogonal in E. coli, PylRS-pylTUCA and PylRS-pylTUCCU pairs are not able to compete efficiently with the endogenous aminoacyl tRNA synthetase-suppressor tRNA pair. At the opal mutation site, tryptophan (Trp) competes with BocK incorporation. Although the PylRS- pylTUCA pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. In the case of AGGA codon, arginine completely outcompetes the incorporation of BocK. This detailed study of tRNAPyl orthogonality provides important insights for developing new bioorthogonal aminoacyl-tRNA synthetase-suppressor tRNA pair.

Systematic studies of nonsense suppression abilities of Methanosarcina mazei pyrrolysyl-tRNA synthetase-tRNA<sup>Pyl</sup> pair in <i>Escherichia coli</i>

247th National Meeting (2014)

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Systematic studies of nonsense suppression abilities of Methanosarcina mazei pyrrolysyl-tRNA synthetase-tRNAPyl pair in Escherichia coli

Systematic studies of nonsense suppression abilities of Methanosarcina mazei pyrrolysyl-tRNA synthetase-tRNA^Pyl pair in Escherichia coli