Fig 1.
Fluorescence intensities of cells expressing GFP6 variant (A and B). GFP6 was expressed in DHF expression hosts outfitted with yPheRS_naph and ytRNAPheAAA in minimal medium supplemented with 18 amino acids, 5.0 μM Phe, 50 μM Trp, and no 2Nal (A); 3 mM 2Nal (B). UUC and UUU codon occupancy by Phe and 2Nal (C and D). Both GFP6 (2UUC) and GFP6 (2UUU) were expressed in DHF expression hosts outfitted with yPheRS_naph and ytRNAPheAAA in minimal medium supplemented with 18 amino acids (25 μg/mL), 50 μM Trp, 3 mM 2Nal, and either 2.5 μM or 5.0 μM Phe. The UUC (C) and UUU (D) codon occupancy by Phe and 2Nal were determined by N-terminal sequencing.
Fig 2.
Extended wobble rules (A). Mutant ytRNAPheAAA recognizing UUU and UUC Phe codons by Watson-Crick (W/C) base pairing and wobble base pairing, respectively (B). Mutant ytRNAPheCAA recognizing UUG Leu codon by W/C base pairing but none of other Leu codons (C).
Fig 3.
Scheme of 2Nal incorporation into multiple UUG Leu codons based on forced ambiguity of the Leu codons.
E. coli leucyl-tRNA synthetase (LeuRS) charges Leu into its cognate tRNALeus containing UAA anticodon (E. coli tRNALeuUAA) and CAA anticodon (E. coli tRNALeuCAA). Leu charged into E. coli tRNALeuUAA is incorporated into multiple UUA Leu codon sites of a target protein. The yPheRS_naph charges 2Nal into ytRNAPhe containing CAA anticodon (ytRNAPheCAA). Then, 2Nal is incorporated into multiple UUG Leu codons. According to the (extended) wobble rules, ytRNAPheCAA and E. coli tRNALeuUAA do not recognize UUA and UUG, respectively. UUG Leu codons can also be recognized by Leu-charged E. coli tRNALeuCAA resulting in partial occupancy of UUG codons by Leu.
Fig 4.
LC-MS chromatogram of tryptic digests of mDHFR.
Peptide 1 (residues 165–180; LCUULCUCPEYPGVLCUCSEVQEEK) contains three Leu residues encoded as CUU and CUC codons. Peptide 2 (residues 54–61; QNLCUGVIMGR) contains a Leu residue encoded as CUG codon. Peptide 3 (residues 62–70; LCUUIEQPELUUGASK) contains two Leu residues encoded as CUU and UUG codons. Peptide 4 (residues 99–105; SLUUGDDALUUAR) contains two Leu residues encoded as UUG and UUA codons. Peptide 4UUA is the same as Peptide 4 except both Leu residues are encoded as UUA codon. Peptide 1; 2; 3; 4; 4UUA variants containing Leu and 2Nal were designated 1L and 1Z; 2L and 2Z; 3L and 3Z; 4L and 4Z; 4UUAL and 4UUAZ, respectively. These peptides were separated by LC and detected by MS. Unmodified mDHFR was synthesized in the absence of 2Nal in a Phe/Leu auxotrophic expression host (A, C, E, and G) in 2xYT media. Modified mDHFRs were synthesized in a Phe/Leu auxotrophic expression host outfitted with ytRNAPheCAA and yPheRS_naph (B, D, F, H, and I). The expression minimal media were supplemented with 17 amino acids (25 μg/mL), 1.25 μM Leu, 50 μM Phe, 50 μM Trp, and 3 mM 2Nal. No 1Z, 2Z, or 4UUAZ was detected by LC-MS analysis.
Fig 5.
Fluorescence intensities of cells expressing GFP3.
GFP3 was expressed in MPC390 expression hosts outfitted with yPheRS_naph and ytRNAPheCAA in minimal medium supplemented with 17 amino acids, 1.25 μM Leu, 5.0 μM Phe, 50 μM Trp, and no 2Nal (A); 3 mM 2Nal (B).
Table 1.
Occupancy of UUU and UAG codons by various amino acids.
Fig 6.
MALDI-TOF spectrum of tryptic digests of mDHFR_38Am.
Peptide Z38 (residues 26–39; NGDLPWPPLRNEZK; Z indicates 2Nal) contains an amber codon at the 38th position. Peptide K38 (residues 26–39; NGDLPWPPLRNEKK; K indicates Lys) contains Lys at the 38th position. Another tryptic digest (residues 85–98; ELKEPPRGAHFLAK) contains Phe.