Spacer-length dependence of programmed -1 or -2 ribosomal frameshifting on a U(6)A heptamer supports a role for messenger RNA (mRNA) tension in frameshifting

Lin, Zhaoru; Gilbert, Robert J. C.; Brierley, Ian

The eluate was concentrated by ethanol precipitation, the mRNA resuspended in water, checked for integrity by agarose gel electrophoresis and quantified by spectrophotometry. Messenger RNAs were translated in nuclease-treated rabbit reticulocyte lysate (RRL; Promega) programmed with $50 mg/ml template mRNA. Typical reactions were of 10 ml volume and composed of 90% (vol/vol) RRL, 20 mM amino acids (lacking methionine) and 0.2 MBq [ 35 S]-methionine. Reactions were incubated for 1 h at 30 C and stopped by the addition of an equal volume of 10 mM ethylenediaminetetraacetic acid (EDTA), 100 mg/ml RNase A followed by incubation at room temperature for 20 min. Samples were prepared for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) by the addition of 10 vol of 2Â Laemmli's sample buffer (34), boiled for 3 min and resolved by SDS-PAGE. Dried gels were exposed to a Cyclone Plus Storage Phosphor Screen (PerkinElmer), the screen scanned using a Typhoon TRIO Variable Mode Imager (GE Healthcare) in storage phosphor autoradiography mode, and bands were quantified using ImageQuant TM TL software (GE Healthcare). Where used, AONs were added to translation reactions on ice at the same time as the mRNA, but were not pre-annealed to the mRNA. The calculations of frameshifting efficiency (%FS) took into account the differential methionine content of the various products and utilized two formulas. RNAs derived from Nco I-cut pFSHIV-AON were translated in RRL (at $50 mg/ml) in the presence of increasing quantities of 25MO or 25OMe. The products were resolved by 15% SDS-PAGE and visualized by autoradiography. In most lanes, an additional product is evident, thought to be derived from ribosomes that enter the +1/À2 frame (+1/À2) or fall off the template (or are permanently stalled) in the vicinity of the annealed AON (drop-off, d.o.). Control translations were also carried out using an mRNA derived from pFScass 5 (Ref. 12; pFScass 5/IBV PK) which contains the minimal IBV frameshift site. The frameshifting efficiency measured for each signal (to the nearest integer) is indicated below the relevant lanes (%FS; see section 'Materials and Methods') and takes into account the number of methionines present in each product (stop, 10; À1 FS, 10; +1/À2/d.o., 11). Due to the close migration of the +1/À2/d.o. product and the stop product in this experiment, the values represent our best efforts for quantification of each class of event. M; molecular size markers. 1 and frameshift 2 products are denoted by Met S , Met FS1 and Met FS2, respectively; the densitometer values for the same products by I S , I FS1 and I FS2 .