In vitro translation

Frameshift reporter plasmids were linearized with FspI and capped run-off transcripts generated using T7 RNA polymerase⁴³. Messenger RNAs were translated in nuclease-treated RRL or WG extracts (Promega) programmed with ∼50 μg ml⁻¹ template mRNA. Typical reactions were of 10 μl volume and composed of 90% (v/v) RRL, 20 μM amino acids (lacking methionine) and 0.2 MBq [³⁵S]-methionine. Reactions were incubated for 1 h at 30 °C and stopped by the addition of an equal volume of 10 mM EDTA, 100 μg ml⁻¹ RNase A followed by incubation at room temperature for 20 min. Proteins were resolved by 12% SDS–PAGE and dried gels were exposed to X-ray film or to a Cyclone Plus Storage Phosphor Screen (PerkinElmer). The screen was scanned using a Typhoon TRIO Variable Mode Imager (GE Healthcare) in storage phosphor autoradiography mode and bands were quantified using ImageQuantTL software (GE Healthcare). PRF efficiencies were calculated as [IFS/MetFS]/[IS/MetS+IFS/MetFS], where the number of methionines in the stop and frameshift products are denoted by MetS and MetFS, respectively, and the densitometry values for the same products are denoted by IS and IFS, respectively. All frameshifting assays were performed at least three times.