FIG. 4.

MBP-nsp10 5′-to-3′ RNA duplex-unwinding activity. Reaction conditions were as described in Materials and Methods with approximately 90 fmol of RNA substrate per reaction. The structures of the substrates are shown schematically with the radiolabeled strands marked by asterisks. The reaction products were separated on nondenaturing, 10 to 20% gradient polyacrylamide gels. The positions of the partially double-stranded substrates (dsRNA) and the displaced monomeric products (ssRNA) are indicated. Lanes: 1, incubation of 5′-RNA2 without protein; 2, heat-denatured 5′RNA2; 3, incubation of 5′-RNA2 with 3 pmol of MBP-nsp10 in the absence of ATP; 4, incubation of 5′-RNA2 with 3 pmol of MBP-nsp10 in the presence of 5 mM ATP; 5, incubation of 5′-RNA2 with 3 pmol of MBP-nsp10-KQ in the presence of 5 mM ATP; 6, incubation of 3′-RNA2 without protein; 7, heat-denatured 3′-RNA2; 8, incubation of 3′-RNA2 with 3 pmol of MBP-nsp10 in the absence of ATP; 9, incubation of 3′-RNA2 with 3 pmol of MBP-nsp10 in the presence of 5 mM ATP; 10, incubation of 3′-RNA2 with 3 pmol of MBP-nsp10-KQ in the presence of 5 mM ATP.