FIG. 5.

Synthesis of RNA from a heteropolymeric template by recombinant norovirus polymerases. Recombinant enzyme (1 μM) was incubated with 1 μg of the 830-nt green fluorescent protein RNA template for 1 h at 30°C in the presence of [α-³²P]UTP; 3 mM MgCl₂ was included in each reaction except that shown in lane 4 (no magnesium). The recombinant protein used in each assay is indicated above the gel (lanes 1 to 6). The synthesized RNA was purified, precipitated in ethanol, dried, heated in glyoxyl sample buffer, and analyzed in a 1% agarose gel. The gel was dried and exposed to film. In lane 7, 1 μg of the green fluorescent protein DNA template was linearized by MluI and transcribed by T7 polymerase in the presence of 10 μCi of [α-³²P]UTP. The sizes of the green fluorescent protein RNA transcript (830 nt) and the RNA dimer (approximately 1,660 nt) produced by the norovirus polymerases are indicated.