Figure 1.

End-to-end template switch by a recombinant BVDV RdRp. (A) Sequence of templates used in these reactions. The arrow denotes the initiation cytidylate. (B) End-to-end template switch with (-)21–3′dd as a function of template concentration. φ is a control reaction performed without RNA. (C) Formation of NRs. At the right of the autoradiogram are schematics of the product from the templates: RecO (hatched box), (-)21–3′dd (open box); (-)15, (black box); (-)12, (gray box). Asterisks (*) identify the NRs. (D) (Top) Schematic of the purified dimeric NR generated from reactions containing donor RecO (boxed) and acceptor Rec1 (gray box with positions of radiolabeled CMPs underlined). Arrows indicate potential sites for RNaseT1 cleavage. (Bottom) Autoradiogram of the results of RNaseT1 digestion with the key components in each reaction indicated above each lane. M denotes a reaction by using donor RecO and acceptor Rec1 similar to one that generated the NR that was purified for analysis in lanes 3–5. Lane 5 contains a darker exposure of lane 4 to show the expected RNaseT1 digestion intermediates. (E) (Top) Schematic of the IR generated from template 14–2 emphasizing the sequence near the switch junction. The sequence whose synthesis is directed by donor is boxed, whereas the sequence from acceptor is shaded. The position of the only radiolabeled CMP is underlined. J1 and J2 are DNA oligonucleotides that should and should not, respectively, stably anneal with the sequence near the switch junction in the IR. The smallest radiolabeled RNA fragment that should remain should be 8 nucleotides. (Bottom) Autoradiogram with results from RNaseH digestions. M contains a reaction similar to the one used to generate the IR that was purified and used in the analysis in lanes 2–4.