Journal of Biological Chemistry
Volume 275, Issue 8, 25 February 2000, Pages 5329-5336
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RNA: STRUCTURE METABOLISM AND CATALYSIS
Poliovirus RNA-dependent RNA Polymerase (3Dpol): ASSEMBLY OF STABLE, ELONGATION-COMPETENT COMPLEXES BY USING A SYMMETRICAL PRIMER-TEMPLATE SUBSTRATE (sym/sub)*

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Detailed studies of the kinetics and mechanism of nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus, 3Dpol, have been limited by the inability to assemble elongation complexes that permit activity to be monitored by extension of end-labeled primers. We have solved this problem by employing a short, symmetrical, heteropolymeric RNA primer-template that we refer to as “sym/sub.” Formation of 3Dpol-sym/sub complexes is slow owing to a slow rate of association (0.1 μm−1 s−1) of 3Dpol and sym/sub and a slow isomerization (0.076 s−1) of the 3Dpol-sym/sub complex that is a prerequisite for catalytic competence of this complex. Complex assembly is stoichiometric under conditions in which competing reactions, such as enzyme inactivation, are eliminated. Inactivation of 3Dpol occurs at a maximal rate of 0.051 s−1 at 22 °C in reaction buffer lacking nucleotide. At this temperature, ATP protects 3Dpol against inactivation with aK 0.5 of 37 μm. Once formed, 3Dpol-sym/sub elongation complexes are stable (t 12 = 2 h at 22 °C) and appear to contain only a single polymerase monomer. In the presence of Mg2+, AMP, 2′-dAMP, and 3′-dAMP are incorporated into sym/sub by 3Dpol at rates of 72, 0.6, and 1 s−1, respectively. After incorporation of AMP, 3Dpol-sym/sub product complexes have a half-life of 8 h at 22 °C. The stability of 3Dpol-sym/sub complexes is temperature-dependent. At 30 °C, there is a 2–8-fold decrease in complex stability. Complex dissociation is the rate-limiting step for primer utilization. 3Dpoldissociates from the end of template at a rate 10-fold faster than from internal positions. The sym/sub system will facilitate mechanistic analysis of 3Dpol and permit a direct kinetic and thermodynamic comparison of the RNA-dependent RNA polymerase to the other classes of nucleic acid polymerases.

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This work was supported in part by Howard Temin Award CA75118 from the NCI and NIAID Grant AI45818 from the National Institutes of Health (both to C. E. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.