Enzymatic and nonenzymatic functions of viral RNA-dependent RNA polymerases within oligomeric arrays

Ability of mutant 3D polymerases bearing active-site mutations and mutations at predicted interfaces to oligomerize and to biochemically complement wild-type polymerase. (A) Rendering of two full-length 3D polymerase molecules (Thompson and Peersen 2004) interacting at predicted Interface I (Hansen et al. 1997). Residues Leu446, Arg455, and Arg456 on the “thumb” side are shown in blue; the “ΔI thumb” mutant polymerase contains L446N, R455A, and R456A mutations. Residues Asp339, Ser341, and Asp349 on the “palm” side of Interface I are shown in pink; the “ΔI palm” mutant polymerase contains D339A, S341A, and D349A mutations. (B) Concentrated wild-type, YGAA ΔI thumb, and YGAA ΔI palm polymerases were diluted to concentrations of 5.6 μM and turbidity was monitored as in Figure 1. (C) The elongation of ³²P-labeled HP1 RNA (lane 1) by 500 nM of wild-type polymerase resulted in 15% template utilization under the conditions of this experiment (lane 2). To this basal level of wild-type polymerase, increasing concentrations of YGAA, YGAA ΔI thumb, and YGAA ΔI palm mutant polymerases were added: 125 nM (lanes 3,9,15), 250 nM (lanes 4,10,16), 500 nM (lanes 4,11,17), 1 μM (lanes 5,12,18), 2 μM (lanes 6,13,19), and 4 μM (lanes 7,14,20). The RNA species present after a 30-min incubation were displayed by gel electrophoresis. (D) The percentage of template elongation from the experiment in C is shown as a function of the ratio of mutant:wild-type polymerase. These experiments were repeated several times; typical results are shown. (E) The elongation of ³²P-labeled HP1 RNA (lane 1) by 500 nM of wild-type polymerase resulted in 14% template utilization under the conditions of this experiment (lane 2). To this basal level of wild-type polymerase, increasing concentrations of YGAA, Δ65, and V33A/F34A mutant polymerases were added: 125 nM (lanes 3,9,15), 250 nM (lanes 4,10,16), 500 nM (lanes 4,11,17), 1 μM (lanes 5,12,18), 2 μM (lanes 6,13,19), and 4 μM (lanes 7,14,20). The RNA species present after a 30-min incubation were displayed by gel electrophoresis. (F) The percentage of template elongation from the experiment in E is shown as a function of the ratio of mutant:wild-type polymerase.