In vitro incorporation of nonnatural amino acids into protein using tRNA^Cys-derived opal, ochre, and amber suppressor tRNAs

Concurrent readthrough of cognate amber and opal stop codons in a truncated mRNA. (A) AQP4-P(98) mRNAs containing stop codons (shown below the gel) were translated in WG extract containing aa-tRNAs (shown above the gel), and proteins were analyzed by SDS-PAGE following RNase treatment. Polypeptides that terminate at residue 44, or readthrough residue 44 but terminate at residue 68, are marked by “–” and “*,” respectively. Polypeptides that end at residue 98 are shown by “**.” Concurrent readthrough efficiency at both codons was 28% based on phosphorimaging (cf. lanes 1 and 12, **). Polypeptides 44, 68, and 98 residues in length contain two, two, and four methionine residues, respectively. The panels shown are taken from the same gel and exposure time. (B,C) NBD-[³H]Lys-tRNA^Lysamb and yeast MBB-[¹⁴C]Cys-tRNA^Cysopl were added individually (B) or together (C) to translation reactions containing apt-12 and programmed with AQP4-P(98) mRNA containing the indicated stop codons (shown below the graphs). The graph shows the amount of [³H]Lys (white bars) or [¹⁴C]Cys (gray bars) incorporated into the translated proteins as measured by hot acid precipitable counts. Values show an average of two experiments.