Figure 1.

Functional moieties in the subgenomic promoter required for initiation of RNA synthesis. (A) Predicted functional groups required for interaction with the BMV RdRp. The sequence shown is proscript −20/13 containing the WT BMV subgenomic promoter directing synthesis of a 13-nt product and serves as the WT control. The subgenomic initiation site is denoted by an arrow with the sequence of the RdRp product shown above. The base moieties predicted to interact with the RdRp by previous mutational studies (8) are indicated below the four nucleotides essential for RNA synthesis. (B) Recognition of the guanylate residue at positions −17 and −11. The bands denoted by ∗ represent terminal transferase labeling of the input template. (C) Recognition of the adenylate at position −14 and the cytidylate at position −13. The structures of the particular base for each position and of the nucleosides in the case of the guanylate at positions −17 and −11 are shown with arrows indicating defined changes in the functional groups mediated by the insertion of various base analogs. Numbers in parenthesis indicate the lane in the autoradiograph containing the RdRp reaction products from proscripts containing the indicated base analog. Lane WT represents the products directed by the −20/13 proscript, and lane ø represents the products of a control reaction with no added template. The reaction products were separated by denaturing PAGE and visualized by autoradiography with their sizes denoted on the side. The predominant RdRp product was 14 nts because of the nontemplated addition of one residue at the 3′ end of the RNA product (8). Accurate initiation was verified by described enzymatic manipulations (8) and comparison to T7-generated size markers. Values listed below the gels represent the percent activity from promoters containing each base analog compared to that from the WT promoter sequence.