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

Incorporation and stability of Cys-tRNAs in RRL and WG extract translation systems. (A) The indicated aa-tRNAs were added to RRL (gray bars) or WG extract (white bars) translation reactions programmed with AQP4-P(98) cDNA containing a cognate cysteine (UGC) or an amber (UAG) codon at residue 44. [¹⁴C]Cys and [¹⁴C]Lys incorporation into protein was measured by liquid scintillation counting of hot acid precipitable counts. Results show mean ± SEM (n ≥ 3). (B–D) Deaminoacylation of Lys-tRNA^Lysamb(•), yeast (▲), E. coli (■), or human (▼) Cys-tRNA^Cys was determined by incubating aa-tRNAs in a mock translation reaction containing RRL (B), WG extract (C), or equivalent buffer (D). At the times indicated, aa-tRNA was precipitated in cold TCA and analyzed by scintillation counting. Counts obtained at t = 0 were used as a reference.