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. 2001 Jul 1;29(13):2715-24.
doi: 10.1093/nar/29.13.2715.

Primer-dependent synthesis by poliovirus RNA-dependent RNA polymerase (3D(pol))

V Rodriguez-Wells  1 , S J Plotch, J J DeStefano
Affiliations

Primer-dependent synthesis by poliovirus RNA-dependent RNA polymerase (3D(pol))

V Rodriguez-Wells et al. Nucleic Acids Res. .

Abstract

Properties of poliovirus RNA-dependent RNA polymerase (3D(pol)) including optimal conditions for primer extension, processivity and the rate of dissociation from primer-template (k(off)) were examined in the presence and absence of viral protein 3AB. Primer-dependent polymerization was examined on templates of 407 or 1499 nt primed such that fully extended products would be 296 or 1388 nt, respectively. Maximal primer extension was achieved with low rNTP concentrations (50-100 microM) using pH 7 and low (<1 mM) MgCl(2) and KCl (<20 mM) concentrations. However, high activity (about half maximal) was also observed with 500 microM rNTPs providing that higher MgCl(2) levels (3-5 mM) were used. The enhancement observed with the former conditions appeared to result from a large increase in the initial level or active enzyme that associated with the primer. 3AB increased the number of extended primers at all conditions with no apparent change in processivity. The k(off) values for the polymerase bound to primer-template were 0.011 +/- 0.005 and 0.037 +/- 0.006 min(-1) (average of four or more experiments +/- SD) in the presence or absence of 3AB, respectively. The decrease in the presence of 3AB suggested an enhancement of polymerase binding or stability. However, binding was tight even without 3AB, consistent with the highly processive (at least several hundred nucleotides) nature of 3D(pol). The results support a mechanism whereby 3AB enhances the ability of 3D(pol) to form a productive complex with the primer-template. Once formed, this complex is very stable resulting in highly processive synthesis.

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Figures

Figure 1
Figure 1
Determination of optimal conditions for primer extension by 3Dpol. Shown are autoradiograms from experiments testing primer extension while changing various reaction parameters (as indicated). The 407 nt template and the 20 nt 5′-32P-labeled primer were used for all experiments. Standard RNA synthesis reaction conditions (see Materials and Methods) were employed except that one parameter (as indicated) was varied in each set of reactions. Fully extended (FL) products are 296 nt in length. Lanes labeled C show reactions in the absence of enzyme. For the MgCl2 titration the concentrations used were (left to right after lane C) 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 mM. For the KCl titration the concentrations used were (left to right after lane C) 1, 4, 10, 20, 40 and 80 mM.
Figure 2
Figure 2
Optimal nucleotide concentrations for reactions with 0.8 or 4 mM MgCl2. The 407 nt template and 20 nt 5′-32P-labeled RNA primer were used for reactions with 0.8 or 4 mM MgCl2 and various amounts of rNTPs. A plot of the relative level of primer extension versus rNTP concentration, [rNTPs], is shown. The highest level of primer extension (0.8 mM MgCl2, 100 µM rNTPs) was set to a value of 1.0 and the levels of primer extension in other reactions are expressed relative to this lane. A repeat of this experiment yielded similar results (data not shown).
Figure 3
Figure 3
Primer extension reactions in the presence or absence of trap with various template concentrations. Shown is an autoradiogram of a primer extension reaction performed in the presence or absence of heparin (trap). The 407 nt template and the 20 nt 5′-32P-labeled primer were used for all experiments. Conditions were as described in Materials and Methods (‘Conditions for reactions to test processivity’) except that the concentration of template was varied as indicated. Lane C, no enzymes added; lane T, trap control (enzyme mixed with heparin trap and rNTPs before mixing with primer-template). The concentrations of primers extended were 0.2, 0.6, 1.7, 5.8, 8.9 and 14.1 nM for reactions without trap and 2.5, 5, 10, 20, 40 and 80 nM template, respectively. The concentrations of primers extended were 0.2, 0.4, 1.1, 2.6, 3.3 and 3.7 nM for reactions with trap and 2.5, 5, 10, 20, 40 and 80 nM template, respectively. All other markings were as described in Figure 1.
Figure 4
Figure 4
Evaluation of reaction conditions on the initial association and turnover of 3Dpol with primer-template in reactions with or without trap. Shown is an autoradiogram of a primer extension reaction performed in the presence or absence of heparin (trap) as indicated. The 407 nt template and the 20 nt 5′-32P-labeled primer were used for all experiments at a concentration of 10 nM template. Conditions were as described in Materials and Methods (‘Conditions for reactions to test processivity’). Enzyme was preincubated with primer-template in 10 µl in a buffer containing 50 mM HEPES pH 7, 10 mM KCl, 5 mM DTT, 0.8 U/µl RNasin and the following: lanes 3 and 4, 0.8 mM MgCl2; lanes 5 and 6, 50 µM rNTPs; lanes 7 and 8, 4.0 mM MgCl2; lanes 9 and 10, 500 µM rNTPs. Preincubations were for 5 min at 30°C. The reactions were initiated by adding 2.5 µl of supplement in the above buffer including the following: lanes 3 and 4, 0.8 mM MgCl2 and 250 µM rNTPs (final concentration 50 µM in reaction); lanes 5 and 6, 50 µM rNTPs and 4.0 mM MgCl2 (final concentration 0.8 mM in reactions); lanes 7 and 8, 4.0 mM MgCl2 and 2.5 mM rNTPs (final concentration 500 µM in reactions); lanes 9 and 10, 500 µM rNTPs and 20 mM MgCl2 (final concentration 4.0 mM in reactions). The supplement also included heparin (trap) as indicated above the lanes. Reactions were continued for 1 h after supplement addition. The percentage of primer extended in the reactions relative to lane 3 (100%) is shown above each lane. All other markings were as described in Figure 3.
Figure 5
Figure 5
Determination of the koff value for 3Dpol on primer-template in the presence or absence of 3AB. Off-rates (koff) were determined as described in Materials and Methods. (A) An autoradiogram from a typical experiment performed in the presence or absence of 3AB (as indicated). The number above each lane indicates the time (min) between the addition of the heparin trap and the rNTPs. Lanes C, no enzymes added; lanes T, trap control (as described in Fig. 4); lanes E, control for primer extension in the absence of heparin. Reactions for lanes C, T and E were incubated for 35 min. Other markings are as described in Figure 2. (B) Plot of a koff experiment: volume (phosphoimager units) versus time from a typical koff experiment is shown. The values for volume were derived from phosphoimager analysis of the above autoradiogram. The curve was fit to an equation for single exponential decay in order to calculate koff (see Materials and Methods). Values for koff were 0.011 ± 0.005 min–1 and 0.037 ± 0.006 min–1 (averages of at least four experiments ± SD), in the presence or absence of 3AB, respectively.
Figure 6
Figure 6
Polymerase 3Dpol is highly processive. Reactions to evaluate the processivity of 3Dpol were performed using the 1499 nt template and a 20 nt 5′-32P-labeled RNA primer as described in Materials and Methods (‘Conditions for reactions to test processivity’). Full extension of the primer would result in a 1388 nt product although the gel system used was unable to resolve products greater than ∼800 nt. An autoradiogram from a primer extension experiment with assays performed using various MgCl2 and KCl concentrations (as indicated) is shown. Some reactions were initiated in the presence of heparin (as indicated) and 3AB was included in some reactions (as indicated). The level of stimulation by 3AB was determined to be ∼3-fold by phosphoimager analysis. Lane M, DNA markers of the indicated sizes in nucleotides; lane C, no enzyme added; lane T, trap control (as described in Fig. 3).
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