STUDIES WITH POLIOVIRUS POLYMERASE 3D(POL) - STIMULATION OF POLY(U) SYNTHESIS IN-VITRO BY PURIFIED POLIOVIRUS PROTEIN 3AB

The synthesis in vitro of poly(U) on a poly(A) template with oligo(dT) (15) primer by poliovirus RNA polymerase 3D(pol) (280 ng/ml) is strong ly stimulated (50-100 fold) by the addition of purified poliovirus pol ypeptide 3AB. The synthesis of product continues linearly with time fo r up to 90 min. The reaction with 3D(pol) alone can be reactivated and similarly enhanced by the addition of 3AB at 30 min of incubation. Op timal stimulation is achieved under conditions where the concentration of 3D(pol) and of template is low, when the molar ratio of 3AB to 3D( pol) is about 100:1 and that of 3AB to poly(A) is about 25:1. In the p resence of 3AB, the yield of product made by 3D(pol) is much increased but its size is unchanged. From a number of basic proteins and peptid es tested, a few were found which also exhibited limited enhancement o f polymerase activity. The stimulatory effect of 3AB is probably relat ed to its ability to bind both the template-primer, poly(A).oligo(dT)( 15), and 3D(pol) (Molla, A., Harris, K. S., Paul, A. V., Shin, S. H., Mugavero, J., and Wimmer, E. J. (1994) J. Biol. Chem. 269, 27015-27020 ). RNA synthesis on purified poliovirus RNA with oligo(dT)(15) primer is enhanced by 3AB about 5-10 fold, and this reaction is highly sensit ive to detergent.