Poliovirus RNA-dependent RNA polymerase (3D(pol)) - Assembly of stable, elongation-competent complexes by using a symmetrical primer-template substrate (sym/sub)

Detailed studies of the kinetics and mechanism of nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus, 3D(pol), hav e been Limited by the inability to assemble elongation complexes that permi t activity to be monitored by extension of end-labeled primers. We have sol ved this problem by employing a short, symmetrical, heteropolymeric RNA pri mer-template that we refer to as "sym/sub." Formation of 3D(pol)-sym/sub co mplexes is slow owing to a slow rate of association (0.1 mu M-1 s(-1)) of 3 D(pol) and sym/sub and a slow isomerization (0.076 s(-1)) of the 3D(pol)-sy m/sub complex that is a prerequisite for catalytic competence of this compl ex. Complex assembly is stoichiometric under conditions in which competing reactions, such as enzyme inactivation, are eliminated. Inactivation of 3D( pol) occurs at a maximal rate of 0.051 s(-1) at 22 degrees C in reaction bu ffer lacking nucleotide. At this temperature, ATP protects 3D(pol) against inactivation with a K-0.5 of 37 mu M. Once formed, 3D(pol)-sym/sub elongati on complexes are stable (t(1/2) = 2 h at 22 degrees C) and appear to contai n only a single polymerase monomer, In the presence of Mg2+, AMP, 2'-dAMP, and 3'-dAMP are incorporated into sym/sub by 3D(pol) at rates of 72, 0.6, a nd 1 s(-1), respectively. After incorporation of AMP, 3D(pol)-sym/sub produ ct complexes have a half-life of 8 h at 22 degrees C. The stability of 3D(p ol)-sym/sub complexes is temperature-dependent. At 30 degrees C, there is a 2-8-fold decrease in complex stability. Complex dissociation is the rate-l imiting step for primer utilization. 3D(pol) dissociates from the end of te mplate at a rate 10-fold faster than from internal positions. The sym/sub s ystem will facilitate mechanistic analysis of 3D(pol) and permit a direct k inetic and thermodynamic comparison of the RNA-dependent RNA polymerase to the other classes of nucleic acid polymerases.