Oligomerization properties of poliovirus 3D polymerase in solution and effects of titration of wild-type and YGAA mutant polymerases into subthreshold concentrations of wild-type polymerase. (A) A ribbon diagram of the three-dimensional structure of the 461-amino acid poliovirus RNA-dependent RNA polymerase is shown with the canonical “thumb,” “fingers,” and “palm” polymerase domains indicated. Asp328 and Asp329 at the active site are shown in yellow; both of these residues were mutated to Ala residues in the YGAA mutant polymerase. Residues Leu446, Arg455, and Arg456, involved in the “thumb” surface contact of a polymerase–polymerase interaction termed Interface I are shown in blue. Residues Asp339, Ser341, and Asp349, involved in the “palm” surface of Interface I are shown in pink; these are difficult to see in this view (see also Fig. 3A). (B) Concentrated wild-type poliovirus polymerase stored in a solution that contained 50% glycerol and 140 mM NaCl was diluted to the concentrations indicated in a solution containing 15% glycerol and 40 mM NaCl. Absorbance measurements were taken every 30 sec over a 10-min time period at 350 nm. (C) Concentrated wild-type and YGAA mutant poliovirus polymerase were diluted to concentrations of 5.6 μM and turbidity was monitored. (D) Electrophoretic mobility of HP1 RNA, a 110-nt RNA derived from the 3′ end of the poliovirus genome with several U residues at its 3′ end for self-priming, is shown following incubation for 30 min with increasing amounts of YGAA mutant polymerase (lanes 2–9, containing 70, 130, 250, 500, 750, 1000, 1500, or 1750 nM YGAA mutant polymerase, respectively). (E) To a preparation of 32P-labeled HP1 RNA (lane 1), various concentrations and mixtures of wild-type and YGAA mutant poliovirus polymerase were added, incubated for 30 min, and the products were displayed (lanes 2–13). Each of lanes 2–13 contained 7.5 nM wild-type polymerase, a concentration too low to support detectable template utilization under these conditions (lanes 2,8). This was supplemented with increasing amounts of wild-type 3D polymerase (lanes 3–7, containing an additional 7.5, 22.5, 55, 117.5, or 242.5 nM wild-type polymerase, respectively) or YGAA mutant polymerase (lanes 9–13) at the same concentrations. (F) The percentage of template elongation is shown as a function of the ratio of added:basal polymerase for both added wild-type and added YGAA mutant polymerase. “WT + WT” and “YGAA + YGAA” show the activities when these polymerases were present without mixing. Data points for turbidity and elongation assays are taken from one representative experiment; at least five experiments were performed for each of the proteins with comparable results. (G) Model for the elongation of RNA within a polymerase lattice. Lateral contacts between polymerase molecules along crystallographically defined Interface I are shown via blue (thumb) and pink (palm) contacts. The contacts (Interface II) that align the Interface I fibers into two-dimensional sheets are not yet known. The RNA template (red) is shown binding to several polymerases along an Interface I fiber, and the nascent RNA (yellow) is shown with its 3′ end in the active site (yellow dots) of a single polymerase molecule. Inactive polymerases mixed into the array are depicted as black molecules.
