Determination of readthrough and misincorporation efficiency in vitro. (A) luc encodes wild-type luciferase; luc-STOP contains a nonsense mutation in position 210 which, upon translation, generates a 23-kDa fragment; and luc* contains a missense mutation in position 218, generating inactive luciferase. (B) The amount of luciferase synthesized in a wild-type lysate primed with luc, luc-STOP, or luc* was determined via quantification of autoradiography. Activity of an aliquot was determined as described in Materials and Methods. As luc-STOP was generated with higher efficiency, only one-third of the sample was loaded onto the gel and applied to the activity assay. The ratio between luciferase activity and intensity (RLU/QL) obtained for luc mRNA was set to 100%.