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. 1999 Mar 11;16(4):190–196. doi: 10.1016/S0167-7799(97)01167-0

Fig. 5.

Fig. 5

Programmed −1 ribosomal frameshift assay strategies. (a) General strategy for directly measuring the efficiency of programmed −1 ribosomal frameshifting. Pictured are the 0-frame control reporter and −1 ribosomal frameshift constructs. Both systems utilize a reporter protein (the lacZ-encoded β-galactosidase, in vivo, or luciferase, in vitro). In the −1 ribosomal frameshift construct, the reporter is cloned downstream of programmed −1 ribosomal frameshift signals and is in the −1 frame with a translational start site. Synthesis of the reporter protein thus requires a programmed ribosomal frameshift event. The 0-frame controls have the reporter genes cloned in frame with the start site and lack the intervening viral frameshift signals. The programmed ribosomal frameshift efficiency is determined by dividing the level of reporter-protein activity produced from the −1 reporters by that produced from the 0-frame control and multiplying by 100%. Translation of the reporters can occur in intact yeast cells or in translationally competent cell extracts. The effects of a candidate compound on both overall translation and on programmed −1 ribosomal frameshifting can be monitored. (b) General strategy for assays in intact yeast cells that detect changes in programmed −1 ribosomal frameshift efficiencies by using a selectable marker. A gene encoding a selectable marker is cloned downstream of, and in the −1 frame with respect to, a translational start site, such that synthesis of the gene product requires a programmed ribosomal frameshift event. Changes in programmed −1 ribosomal frameshift efficiencies are detected by monitoring the ability of cells to grow (positive selection) or not (negative selection) in the presence of a candidate compound. (c) Bicistronic constructs for measuring changes in programmed −1 ribosomal frameshifting efficiencies. Reporter-gene 1 constitutes the 0-frame control and can be used to monitor the effect of a compound on overall translation. Translation of reporter-gene 2 requires a programmed ribosomal frameshift event. Changes in programmed −1 ribosomal frameshift efficiencies are detected by monitoring for changes in the ratios between the activities of the two reporter proteins.