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-tRNAPyl 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 tRNAPyl 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.