FIG. 7.

Requirements for plus-strand RNA synthesis. (A) The predicted secondary structure of B2(−)46G with the stems (A1 and A2) and loops (L1 and L2) indicated by brackets. The initiation cytidylate is denoted by an arrow. (B) Analysis of the sequence in the predicted stem-loop region. The templates used in the specified reactions are indicated at the top of the autoradiogram. Endscripts that have a +1 are indicated by +, while endscripts with changes of the initiation cytidylate to a guanylate are indicated by −. The sizes of the RdRp products are denoted on the right. The amounts of plus-strand synthesis directed by B2(−)Δ3–11 and B2(−)Δ17–26 were 200 and 70%, respectively, compared to synthesis directed by B2(−)46G after correcting for the number of CMP units incorporated. The results presented are an average from three independent trials. (C) Additional analysis of sequences required for efficient RNA synthesis. The templates used and whether they can direct the initiation of RNA synthesis (+ or −) are indicated at the top of the autoradiogram. The sizes of the RNA products are denoted on the right of the autoradiogram. The amounts of synthesis, after adjusting to the number of radiolabelled CMP units incorporated, from B2(−)26G, B2(−)26TV, B2(−)22G, and B2(−)16G were 100, 17, 22, and 5% respectively. (D) Alignment of the sequences in B2(−)26G, B2(−)Δ3–11 (deletion of A2 stem region), B2(−)Δ17–26 (deletion of L1 loop region), and B2(−)26TV (transversion of nt 17 to 24). The two guanylates as well as the two adenylates putatively required for efficient synthesis are shown in bold type.