Nonsense and sense suppression abilities of original and derivative Methanosarcina mazei pyrrolysyl-tRNA synthetase-tRNA(Pyl) 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-tRNA(Pyl) pairs and cross recognition between nonsense codons and various tRNA(Pyl) anticodons in the Escherichia coli BL21(DE3) cell strain are reported. tRNA(CUA)(Pyl) 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, N(ε)-tert-butoxycarbonyl-L-lysine (BocK). Similar to tRNA(CUA)(Pyl), tRNA(UUA)(Pyl) is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. Although tRNA(UUA)(Pyl) is expected to recognize a UAG codon based on the wobble hypothesis, the PylRS-tRNA(UUA)(Pyl) 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-tRNA(UCA)(Pyl) pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. tRNA(CCU)(Pyl) fails to deliver BocK at an AGG codon when co-expressed with PylRS in E. coli.

Figure 1. The structures of Pyl, BocK and AzF.

Figure 2. Suppression of amber, opal, and ochre mutations at N134 of sfGFP by their corresponding PylRS-tRNA^Pyl pairs in the absence and presence of BocK.

(A) Proteins shown in the gel represent their real relative sfGFP expression levels. Lanes 1 and 2 were transformed with pETtrio-PylT(CUA)-PylRS-sfGFP134TAG; lanes 3 and 4 were transformed with pETtrio-PylT(UUA)-PylRS-sfGFP134TAA; lanes 5 and 6 were transformed with pETtrio-PylT(UCA)-PylRS-sfGFP134TGA. ESI-MS spectra of sfGFP expressed in cells (B1) transformed with pETtrio-PylT(CUA)-PylRS-sfGFP134TAG and grown in the presence of 5 mM BocK, (B2) transformed with pETtrio-PylT(UUA)-PylRS-sfGFP134TAA and grown in the presence of 5 mM BocK, (B3) transformed with pETtrio-PylT(UCA)-PylRS-sfGFP134TGA and grown in the absence of BocK, and (B4) transformed with pETtrio-PylT(UCA)-PylRS-sfGFP134TGA and grown in the presence of 5 mM BocK.

Figure 3. Suppression of an opal mutation at S2 of sfGFP by the PylRS- pair.

(A) Expression of sfGFP with an opal mutation. Lanes 1 and 2 were transformed with pETtrio-pylT(UCA)-sfGFP134TGA and grown in the absence or presence of 5 mM BocK; lanes 3 and 4 were transformed with pETtrio-pylT(UCA)-sfGFP2TGA and grown in the absence or presence of 5 mM BocK. Each protein shown in the gel represents their real relative expression levels. ESI-MS of sfGFP expressed in cells transformed with pETtrio-pylT(UCA)-sfGFP2TGA and grown in the (B1) absence or (B2) presence of 5 mM BocK.

Figure 4. Suppression of an opal mutation at N134 of sfGFP at different conditions.

(A) Proteins shown in the gel represent their real relative expression levels. Lane 1 was transformed with pET-sfGFP134TGA; lanes 2 and 3 were transformed with pET-pylT(UCA)-sfGFP134TGA and grown in the absence or presence of 5 mM BocK. (B) ESI-MS of sfGFP expressed in cells transformed with pETtrio-sfGFP134TGA.

Figure 5. Suppression of an opal mutation at N134 of sfGFP by different variants.

(A) Proteins shown in the gel represent their real relative expression levels. Lanes 1 and 2 were transformed with pETtrio-pylT(UCA)G73C-sfGFP134TGA and grown in the absence or presence of 5 mM BocK; lanes 3 and 4 were transformed with pETtrio-pylT(UCA)G73A-sfGFP134TGA and grown in the absence or presence of 5 mM BocK; lanes 5 and 6 were transformed with pETtrio-pylT(UCA)G73U-sfGFP134TGA and grown in the absence or presence of 5 mM BocK; lanes 7 and 8 were transformed with pETtrio-pylT(UCA)-sfGFP134TGA and grown in the absence or presence of 5 mM BocK. The ESI-MS analysis of sfGFP expressed in cells transformed with pETtrio-pylT(UCA)G73U-sfGFP134TGA and grown in the (B1) absence or (B2) presence of 5 mM BocK.

Figure 6. Cross recognitions between different anticodons of tRNA^Pyl and nonsense mutations at N134 of sfGFP.

Cells were transformed with pETtrio-PylT(NNN)-PylRS-sfGFP134N’N’N’ and grown in the presence of 5 mM BocK (NNN and N’N’N’ denote anticodons and codons specified in the figure). Proteins shown in the gel represent their real relative expression levels.

Figure 7. Expression of sfGFP in cells transformed with pETtrio-PylT(UUA)-PylRS-sfGFP134TAG.

(A) Cells grown in 2YT medium supplemented without or with BocK. ESI-MS of sfGFP expressed in the (B1) absence or (B2) presence of 5 mM BocK.

Figure 8. Expression of sfGFP in cells transformed with pETtrio-PylT(UUA)-PylRS-sfGFP134TAG and pEVOL-AzFRS.

(A)Cells were grown in 2YT medium supplemented with different combinations of NAAs. ESI-MS of sfGFP expressed in the(B1) absence of both AzF and BocK; (B2) presence of 1 mM AzF; (B3) presence of 5 mM BocK; and (B4) presence of both 1 mM AzF and 5 mM BocK.

Figure 9. Suppression of an AGG mutation at S2 of sfGFP by.

(A) Expression of sfGFP in cells transformed with pETtrio-pylT(CCU)-sfGFP2AGG and grown in the absence or presence of 5 mM BocK. (B) The ESI-MS analysis of sfGFP expressed in the presence of 5 mM BocK.