Rational design of a synthetic mammalian riboswitch as a ligand-responsive -1 ribosomal frame-shifting stimulator

Lin, Ya-Hui; Chang, Kung-Yao

A rabbit reticulocyte lysate system (Ambion) was used to generate shifted and non-shifted protein products. Capped reporter mRNAs were in vitro transcribed by T7 RNA polymerase supplemented with a methylated cap analogue (Epicentre) in the reaction. The purified capped reporter mRNA (100 ng) was used in a 5 l in vitro translation reaction containing 2.55 l rabbit reticulocyte lysate, 0.25 l of translation buffer, 0.1 l of RNase inhibitor (40 U/l), 0.1 l of 10 Ci/ l [ 35 S]-labeled methionine (NEN) and 1 l of theophylline of varied concentrations. The reaction mixtures were incubated at 30 • C for 1.5 h, and then loaded into 12% sodium dodecylsulphate-polyacrylamide (SDS-PAGE) gels for electrophoresis analysis. The gels were exposed to a phosphorimager screen after drying and the radioactivity of translated products analyzed. The radioactivity of protein products was calibrated with the methionine content of each protein product. Estimated frameshifting efficiency was calculated by dividing calibrated radioactive intensity of full-length shifted protein products by the sum of calibrated radioactive intensity of full-length shifted and non-shifted protein products. Because translation products due to ribosome drop-off in the −1 frame (radioactivity detectable or non-detectable) were difficult for accurate measurement as well as methionine calibration, they were not included in the calculation and would lead to underestimation of frameshifting efficiency. By assuming similar extent of drop-off tendency, we present the effect of theophylline on radioactivity-based −1 PRF activity in terms of relative −1 PRF so that the ribosome drop-off effect can be filtered out (23) . Dual luciferase activity was measured from in vitro translation reactions (without addition of labeled methionine) or lysates of reporter transfected cells for frameshifting efficiency calculations by Dual Luciferase TM reporter assay (Promega) following manufacturer's instructions on a CHAMELEON TM multi-label plate reader (HIDEX). Dual-luciferase based frameshifting efficiency of a specific construct was calculated according to previously described procedures (23) by comparison with a corresponding read-through control assuming that similar extent of ribosome drop-off occured during translation. Each read-through control has the TTTAAAC slippery sequence replaced by a CTTAAGAA sequence that disrupts the slippery site and shifts the reading frame to −1 frame by one extra nucleotide insertion. Unless specified, these readthrough controls (listed in Supplementary Table S1) were used for calibration in frameshifting activity calculation.