FIG. 6.

Effect of the UGACC→UGAACC mutation on the efficiency of minus-strand synthesis. SA11 gene 5 mRNAs were prepared that ended with the 3′ consensus sequence UGACC (GACC) or the atypical sequence UGAACC (GAACC) and used as templates for the synthesis of minus-strand RNA in the open-core replication system. Reaction mixtures contained various amounts of template RNA (A) or open cores (B) and included [³²P]UTP to radiolabel RNA products. ³²P-labeled dsRNA products were detected by SDS-PAGE and autoradiography, and the relative levels of dsRNA products were quantified with a PhosphorImager. The percent GAACC/GACC was calculated by dividing the amount of dsRNA product made in reactions containing template RNA ending with UGAACC by the amount of that ending with UGACC and multiplying the result by 100.