FIG. 2.

The NRS alone can stimulate RSV polyadenylation. The indicated RSV polyadenylation substrates (see Fig. 1) containing a 3′ ss (A) and/or the NRS (B) were uniformly labeled with ³²P and incubated in HeLa nuclear extract for the times (in minutes) indicated above each lane. Polyadenylation appears as an upward smear. Samples were subjected to electrophoresis on a 6% 8 M urea polyacrylamide gel, and images were obtained with a PhosphorImager. Ad3′-RSV and src3′-RSV samples (A) and NRS-src3′-RSV samples (B) were run ∼1 h longer to allow adequate separation of products. The images are representative of at least three independent repeats. M, ³²P-end-labeled pBR322/MspI markers, the sizes of which are indicated at the left and right. The results of quantitation of polyadenylation at 30 min are the following: (A) SVL, 32%; RSV, 2%; Ad3′-RSV, 8%; and src3′-RSV, 4%; (B) SVL, 47%; RSV, 6%; NRS-RSV, 63%; and NRS-src3′-RSV, 7%.