FIGURE 4.

Clitocine incorporation causes readthrough. (A) Graph showing that incorporation of clitocine results in readthrough of the Luc-190-UGA mRNA. Luc-190-UGA was transcribed by T7 RNA polymerase with varying ratios of clitocine-TP to ATP (x-axis). The resultant RNA was purified, translated using rabbit reticulocyte lysate (RRL), and luciferase enzyme activity was determined (y-axis). Error bars represent standard deviation of two determinations. (B) The Luc-190-UGA cDNA was used in coupled transcription–translation reactions with varying ratios of clitocine-TP and ATP (x-axis). Luciferase activity in the reactions is indicated on the y-axis. Error bars represent the standard deviation of two determinations. (C) Schematic representation of peptides produced from chemically synthesized RNAs (see Materials and Methods). During translation in the RRL system, Transcend tRNA was used to label lysine residues with biotin, and [³H]-leucine was used to label leucine residues with tritium. X indicates different triplet codons synthesized into the three RNAs used in panel D. (D) The indicated RNAs were translated in RRL and labeled as described above. After binding of the resultant peptides to streptavidin magnetic beads, the beads were washed, scintillation fluid was added, and the amount of tritium was quantified (y-axis). Error bars represent the standard deviation of the mean for three determinations.