FIGURE 1.

Nucleotide addition by PV RdRp. A, PV RdRp is a prototypical RdRp with fingers, palm, and thumb subdomains. Shown is the WT PV RdRp-RNA complex (PDB code 3OL6) (19). The conserved structural motifs are essential for faithful, efficient, nucleotide addition and are color-coded as follows: motif A, dark green; motif B, tan; motif C, dark cyan; motif D, blue; motif E, golden yellow; motif F, red; and motif G, light green. Gly-64 and His-273 are residues remote from the active site that increase or decrease PV RdRp fidelity, respectively (5, 6, 8). B, nucleotide addition has been shown to require at least five kinetically resolvable steps: step 1, binding of nucleotide to the RdRp-RNA complex; step 2, conformational change to produce a catalytically competent state; step 3, chemistry; step 4, conformational change to permit translocation; step 5, pyrophosphate release (16). Step 2 is a fidelity checkpoint, the physical basis of which is only partially understood (17). C, indicated is the current understanding of nucleotide addition of PV RdRp gleaned from crystal structures (PDB codes 1RA6, 3OL6, and 3OL7) (18, 19). Shown are the conserved residues of conserved structural motifs, colored by motif as indicated for A, that feature prominently in the studies reported herein. In the free enzyme, the conformation of the residues shown will not support binding of RNA or NTP. The binary complex shows substantial rearrangement of most residues; however, NTP binding will still be inhibited by the steric clashes with Asp-238. The closest structure to the catalytic state solved is for a complex after nucleotide incorporation. Here it is clear that every residue shown has had to undergo a conformational change relative to the binary complex for nucleotide addition to occur. Filling in the gaps in going from binary complex to post-incorporation complex is the focus of this study.