FIG. 3.

Synthesis of pRNA by the mutant P47 proteins with dideoxynucleotides to arrest chain extension. P47 and P47-K241R proteins were incubated under the normal pRNA synthesis conditions, except that dideoxynucleotides replaced specific rNTPs in order to terminate chain extension at different locations on the template. (A) Template sequence showing where the pRNA chain synthesized on G4oric would be terminated by ddTTP and ddCTP. (B) pRNA synthesized by P47 and P47-K241R in the presence of all four NTPs (lanes 2 and 5), and when ddTTP (ddT) (lanes 3 and 6) and ddCTP (ddC) (lanes 4 and 7) were added to the incubation mixture. pRNA products were separated on a 23% polyacrylamide gel (acrylamide-to-bisacrylamide ratio of 8:1) containing 7 M urea. The gel was run so that the free [α-³²P]GTP remained in the gel. Two autoradiographic exposures (15 s and 30 min, respectively) were necessary to visualize both free [α-³²P]GTP and the dimer or trimer pRNA.