Assembly, purification, and pre-steady-state kinetic analysis of active RNA-dependent RNA polymerase elongation complex

Abstract

NS5B is the RNA-dependent RNA polymerase responsible for replicating hepatitis C virus (HCV) genomic RNA. Despite more than a decade of work, the formation of a highly active NS5B polymerase·RNA complex suitable for mechanistic and structural studies has remained elusive. Here, we report that through a novel way of optimizing initiation conditions, we were able to generate a productive NS5B·primer·template elongation complex stalled after formation of a 9-nucleotide primer. In contrast to previous reports of very low proportions of active NS5B, we observed that under optimized conditions up to 65% of NS5B could be converted into active elongation complexes. The elongation complex was extremely stable, allowing purification away from excess nucleotide and abortive initiation products so that the purified complex was suitable for pre-steady-state kinetic analyses of polymerase activity. Single turnover kinetic studies showed that CTP is incorporated with apparent K(d) and k(pol) values of 39 ± 3 μM and 16 ± 1 s(-1), respectively, giving a specificity constant of k(pol)/K(d) of 0.41 μM(-1) s(-1). The kinetics of multiple nucleotide incorporation during processive elongation also were determined. This work establishes a novel way to generate a highly active elongation complex of the medically important NS5B polymerase for structural and functional studies.

FIGURE 1.

pGG primer extension and pause assay. A, a scheme depicting the reaction setup and the 20-mer RNA template used in all assays in this study. B, a time course of product formation during an extension and pause assay. The reaction was started by mixing 20 μm pGG, 20 μm template, 25 μm ATP, and 25 μm UTP with 12 μm HCV NS5B. An aliquot from the reaction was mixed with quench solution at each indicated time point; in the meantime, another aliquot of the reaction was reacted with 50 μm CTP and quenched after 20 s (indicated as +C). C, extension and pause assays at various concentrations of NS5B. Each reaction was started with NS5B (2, 4, 6, 8, 10, and 12 μm), 20 μm pGG, 20 μm template, 25 μm ATP, and 25 μm UTP. Aliquots from each reaction were mixed with quench solution at the time points indicated. Concentration of the elongation complex (9-mer and 10-mer) was plotted against NS5B concentration at each reaction time point. For each reaction time, the data were fit to a linear function with the slope representing the fraction of input NS5B assembled into the elongation complex.

FIGURE 2.

Active site titration of HCV NS5B. A, extension and pause reactions were started with 12 μm NS5B, 500 μm ATP, 500 μm UTP, 50 μm 3′-dCTP and pGG/20-mer at various concentrations (5, 10, 20, 40, 60, 80, 100 μm as indicated). The reactions were quenched after 24 h. B, the concentration of elongation complex (10-mer) was plotted against pGG concentration. The data were fit to a quadratic equation, yielding an apparent K_d of 3.8 ± 1.4 μm and an amplitude of 7.8 ± 0.22 μm. Each data point on the plot was the average of four independent experiments, and the error bar was S.D.

FIGURE 3.

Purification of the elongation complex by centrifugation. An extension and pause reaction with 12 μm NS5B, 12.5 μm UTP, 25 μm ATP, and 10 μm pGG/20-mer was run for 2 h, followed by centrifugation at 16,000 × g for 5 min. Lane 1, pGG primer; lane 2, the 2-h reaction before spin; lane 3, one aliquot of the 2-h reaction was reacted with 50 μm CTP for 20 s; lanes 4 and 5, the supernatant (Sup) and its reaction with 50 μm CTP for 20 s; lanes 6 and 7, the resuspended pellet and its reaction with 50 μm CTP for 20 s.

FIGURE 4.

Solubility and stability of the elongation complex. A, solubility of the elongation complex at various NaCl concentrations. The pellet containing the elongation complex after centrifugation was resuspended in the reaction buffer with NaCl at various concentrations (0 ∼ 350 mm). After a 30-min incubation, the samples were spun at 16,000 × g for 5 min. The percentage of elongation complex distributed in the supernatant or the pellet was analyzed by sequencing gel. The percentage of elongation complex in the supernatant (soluble EC) was plotted against NaCl concentration. B, stability of the elongation complex at various NaCl concentrations. The pellet-containing elongation complex was resuspended in reaction buffer with NaCl at various concentrations and was incubated for 14.3 h. The activities of the elongation complex preincubation and postincubation were measured by reacting the elongation complex with 50 μm CTP for 20 s. The percentage of the 10-mer product versus NaCl concentration was shown. C, stability of the elongation complex in the reaction buffer with heparin. The pellet containing elongation complex was resuspended in the reaction buffer with 150 mm NaCl and 0.2 mg/ml heparin and incubated for various time intervals. The remaining activity of the elongation complex was measured by reacting an aliquot at each incubation time point with 50 μm CTP for 20 s. The percentage of the 10-mer product versus incubation time was shown.

FIGURE 5.

K_d and k_pol of single nucleotide incorporation catalyzed by HCV NS5B in the elongation mode. A, time courses of CTP incorporation opposite G in the template at various CTP concentrations (3.1, 6.25, 12.5, 25, 50, 100, and 200 μm) were obtained by rapid quench-flow assays. Data were fit to the mechanism in Scheme 1 using KinTek Global Kinetic Explorer. The solid lines were the fitted lines. The fitted parameters, K_d (equilibrium dissociation constant for nucleotide binding) and k_pol (nucleotide incorporation rate), are 39 ± 3 μm and 16 ± 1 s⁻¹, respectively. B, time courses of GTP incorporation opposite C in the template at various GTP concentrations (3.1, 6.25, 12.5, 25, 50, 100, and 200 μm). The K_d is 22 ± 2 μm, and the k_pol is 2.1 ± 0.1 s⁻¹. Each data set shown is the representative of three repeats, and the K_d and k_pol were reported as mean ± S.D. from the repeats.

FIGURE 6.

Processive nucleotide incorporation by HCV NS5B in the elongation mode. A, a sequencing gel showing the time course of multiple nucleotide incorporation. The reaction was conducted by mixing the elongation complex, NS5B·9-mer·20-mer (0.3 μm), with ATP, UTP, GTP, and CTP (400 μm each) in the reaction buffer and quenched at indicated time intervals. B, the time courses of product formation were analyzed globally by fitting to the mechanism in Scheme 2. The data set shown was a representative from three repeats, with the solid lines indicating the best fit. The fitted rate for each step was listed in Scheme 2 and C. C, a bar graph showing rate (±S.E.) for each nucleotide incorporation during processive replication. The average of the rates from 11 steps was 4 s⁻¹.