FIGURE 8.

Mapping of the primer-dependent initiation sites of c84 and its mutants. A, the 3′-end sequences of c84wt, c84(U4A), c84(U1A,U4A), and c83 are indicated in boldface type, and the initiation sites of the di- and trinucleotide primers are indicated by arrows. U at the fourth position (−4) from the 3′-end is surrounded by a square. The mutated sequences are underlined. The added G and de novo synthesized ApG are shown in italic type. B, c84wt was incubated with a 0.1 mm concentration of the dinucleotide primer AG, AA, GA, GU, or UA in 0.5 mm ATP, CTP, or GTP and 50 mm [α-³²P]UTP. C, c84(U4A) was incubated without (de novo) or with 0.1 mm AG in 0.5 mm ATP, GTP, or UTP and 50 mm [α-³²P]CTP or in 0.5 mm ATP, CTP, or GTP and 50 mm [α-³²P]UTP. D, c84(U4A), c84(U1A,U4A), and c83 were incubated without (de novo) or with a 0.1 mm concentration of the primer AG, AGU, or UU in 0.5 mm ATP, GTP, or UTP and 50 mm [α-³²P]CTP. c84wt transcribed with AG in 0.5 mm ATP, CTP, or GTP and 50 mm [α-³²P]UTP was used as the size marker (c84: AG). The sizes of the RNA products are indicated on the left of the PAGE. Products are marked with their sizes in the PAGE.