FIG. 3.

Copying in vitro of minus-strand [(-)strand] RNA3 templates with BMV replicase. (A) Diagram of RW-IDrev1 RNA template. The positive-sense RNA initiation sequence [(+)promoter] is represented by a solid bar, with flanking nucleotide positions corresponding to those of the wt RNA3 shown below. The sequences complementary to the 3a and CP ORFs are shown as open boxes, and the oligo(U)-containing subgenomic promoter as a shaded box. The sgRNA3a molecule (lower line) carries the 3′-oligo(A) tail that is represented by a gray box. The locations of the RNA linker and those of both DNA primers used for PCR amplification are shown as a black line and arrows, respectively. (B) Electrophoretic analysis of RNA products after in vitro copying of RW-IDrev1 RNA3. The RNA (1 μg) was copied in the RdRp assay reaction (see Materials and Methods) by using a BMV replicase preparation, and the radioactive products were separated by electrophoresis in a 4% polyacrylamide-12 M urea denaturing gel (lane 1). Besides a full-length copy of the input RW-IDrev1 RNA3 template, shorter products of the premature termination reaction at the oligo(U) tract (1,400 nt) and subgenomic sgRNA4 (800 nt) are visible. Lanes M1 and M2 show separate migrations of radioactive in vitro-transcribed RNAs used as size standards: the 800-nt RNA transcript the size of sgRNA4 and the 1,221-nt marker corresponding to the size of sgRNA3a. Repl., BMV replicase. (C) The effect of ATP on the copying of RW-IDrev1 RNA3. The copying reactions at increased ATP concentrations (indicated at the bottom) and analysis of the products were performed as described for panel B.