FIG 1.
In vitro polymerase activity of EMCV 3Dpol. (A to D) In vitro EMCV 3Dpol elongation activity measuring incorporation of [3H]UTP using poly(rA)/dT15 as the template-primer. (A to C) Elongation activity of EMCV 3Dpol while varying cofactor MgCl2/MnCl2, UTP, and glycerol concentrations and the type of buffer. The initial velocity of the reaction (Vi) represents the polymerase elongation activity. The Vi observed with the starting conditions for the optimization was set to 100%. (D) Elongation activity of EMCV 3Dpol using the optimized conditions. (E) EMCV 3Dpol elongation activity as visualized by denaturing PAGE. The dT15 primer is elongated with ([32P])UTP using a poly(rA) template. (F) VPg uridylylation assay. In the presence of a poly(rA) template, [32P]UTP is covalently coupled to the primer VPg by EMCV 3Dpol. Reaction products were analyzed using Tris-tricine-SDS-PAGE.