FIG. 2.

RNA LE19 and the products that could form by de novo initiation and primer extension. (A) LE19 can form a stem-loop structure that mimics the structure found to be conducive for RNA synthesis (structure on the left) (see reference 14). Two molecules of LE19 could also anneal to allow primer extension from the 3′-most nucleotide of each RNA (structure on the right). In RNA LE21, two nucleotides, A and U, are inserted near the 3′ end (denoted with triangles), increasing the number of base pairs in the heterodimer from six to eight, thus favoring primer extension. The 19-nt product from de novo initiation and the 32-nt product made from primer extension are shown in the boxes. The expected RNase T₁ cleavage sites from these two RNAs are shown with arrows, and the radiolabeled products of 18 nt are identified. (B) Autoradiogram of the products by the HCV RdRp Δ21 from LE19 and LE19P, a modified version of LE19 containing a 3′ puromycin, and LE21. The reactions were performed with (+) or without (−) 200 μM GTP. Reactions in lanes identified by “T1” were treated with RNase T₁, while those in lanes denoted with “U” were untreated. The number of nucleotides in the RdRp products is shown on the sides of the autoradiogram. The ratio of de novo initiation to primer extension (PE) RNA products obtained with LE19 and LE21 is given at the bottom of the autoradiogram. The lengths were identified by comparison to multimeric products of recombination from the BVDV RdRp using templates of 13, 14, 15, and 20 nt (15). (C) The structures of LE19 and LE21 in solution, as analyzed by nondenaturing gel electrophoresis. The analyses used 20% polyacrylamide gel that lacked urea. Two gels were run in parallel, one at 20°C and one at 4°C. All RNAs were in a buffer that contained 100 mM KCl and a 2 mM concentration of the divalent metal indicated above the stained gel. A faint band indicative of higher-order interactions (*) is observed in the gel electrophoresed at 4°C.