The capped reporter mRNAs were prepared by the mMESSAGE mMACHINE high-yield capped RNA transcription kit (Ambion) by following the manufacturer's instructions. The reticulocyte lysate system (Progema) was used to generate the shifted and non-shifted protein products. In each assay, a total of 5 ml reaction containing 250 ng of capped reporter mRNA, 2.5 ml of reticulocyte lysate and 0.2 ml of 10mCi/ml 35 S-labeled methionine (NEN) was incubated at 30 C for 1.5 h. The samples were then resolved by 12% SDS polyacrylamide gels and exposed to phosphorimager screen for quantification after drying. The À1 PRF efficiency was calculated by dividing the counts of the shifted product by the sum of the counts for both shifted and non-shifted products, with calibration of the methionine residue number in each product.