FIG 5.

Primer extension assay. nsp8 activity assays were performed using a primer/template substrate (KR01/JTR1) described in an earlier study (24) and a derivative of this substrate in which the bottom strand was 3′ biotinylated (KR01/JTR1-b). (A) Primer/template RNA hybrids used in the experiment. Bio, RNA 3′ biotinylation. (B) Assays were performed in reaction buffer supplemented with 2 μM nsp8, 50 mM NaCl, 4 mM MgCl₂, 50 μM ATP and/or GTP, 0.17 μM [α-³²P]ATP (*ATP), or [α-³²P]GTP (*GTP), as indicated, and 1 μM the indicated RNA substrate. The reaction mixtures were incubated at 30°C for 60 min. Products were resolved in a TBE-buffered 12% polyacrylamide–7 M urea gel and visualized by phosphorimaging. Sizes (in nucleotides) of 5′-³²P-labeled marker RNAs (lane M) are indicated to the left. Lane –, activity assay performed with KR01/JTR1 in the absence of nsp8.