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. tRNACUAPyl 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 totRNACUAPyl, tRNAUUAPyl is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. Although tRNAUUAPyl is expected to recognize a UAG codon based on the wobble hypothesis, the PylRS-tRNAUUAPyl 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-tRNAUCAPyl pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. tRNACCUPyl fails to deliver BocK at an AGG codon when co-expressed with PylRS in E. coli. © 2013 Odoi et al.