Fidelity of Nucleotide Incorporation by the RNA-Dependent RNA Polymerase from Poliovirus
Using poliovirus (PV) and its RNA-dependent RNA polymerase (RdRp) as our primary model system, we have advanced knowledge fundamental to the chemistry and fidelity of nucleotide addition by nucleic acid polymerase. Two fidelity checkpoints exist prior to nucleotide addition. The first toggles the enzyme between a nucleotide binding-occluded state and a nucleotide binding-competent state. The second represents an ensemble of conformational states of conserved structural motifs that permits
... ion of the incoming nucleotide in a state competent for phosphoryl transfer long enough for chemistry to occur. Nucleophilic attack of the alphaphosphorous atom of the incoming nucleotide produces a pentavalent transition state, collapse of which is facilitated by protonation of the pyrophosphate leaving group by a general acid. All of the relevant conformational states of the enzyme are controlled by a network of interacting residues that permits remote-site residues to control active-site function. The current state of the art for PV RdRp enzymology is such that mechanisms governing fidelity of this enzyme can now be targeted genetically and chemically for development of attenuated viruses and antiviral agents, respectively. Application of the knowledge obtained with the PV RdRp to the development of vaccines and antivirals for emerging RNA viruses represents an important goal for the future. have established the RdRp from poliovirus (PV), as a model system for understanding the chemical, kinetic, thermodynamic, structural, and dynamical mechanisms employed by this class of polymerases. Studies presented in subsequent sections of this chapter will hopefully leave you convinced that the PV RdRp system is not only a useful model for the RdRp but for all classes of polymerases. Universal and Adapted Features Revealed by Polymerase Structures Crystal structures have been solved for single-subunit polymerases representing all four classes of polymerases         . In general, the overall topology of polymerases resembles a cupped, right hand with fingers, palm, and thumb subdomains. The RdRp contains an extension of the fingers, the so-called fingertips, that interacts with the thumb subdomain, leading to a completely encircled active site in this class of polymerases (Fig. 1A) . The palm subdomain is the most conserved subdomain of polymerases and can be divided into several conserved structural motifs. In the case of the RdRp, seven conserved structural motifs exist: A-G (Fig. 1A) . Motifs A and C contain conserved aspartic acid residues involved in binding the divalent cation cofactors required for nucleotidyl transfer (Fig. 1B) . Motif A extends from the (deoxy)ribose-binding pocket to the catalytic center (Fig. 1B) . Elements of this motif interacting with the triphosphate moiety and divalent cation superimpose exactly in different polymerases. Elements of this motif interacting with the sugar have evolved to sense the appropriate sugar configuration, thus linking the NTP-binding site to the catalytic site. Motif B contains residues that interact with the (deoxy)ribose moiety of the nucleotide substrate. Motif D was thought to provide structural integrity to the palm subdomain; however, the studies described herein have altered substantially this long-standing view by showing a role for this motif in the chemistry of nucleotidyl transfer. The remaining motifs are unique to RdRps, although some overlap with RTs also exists. Motif E provides interaction between the enzyme and primer. Motif F interacts with the triphosphate moiety of NTP and most likely functions at the earliest step of NTP binding. Motif G has no known function. Structural information is also available on polymerase elongation complexes [11,      . Upon NTP binding, the fingers subdomain often moves relative to the thumb sub-domain, causing an open-to-closed transition that has been considered a rate-limiting step in the nucleotide-addition cycle. This view is now being reconsidered [22,23]. All Polymerases Employ a Two-Metal-Ion Mechanism for Catalysis Given the conserved nature of the catalytic center of polymerases, the chemical mechanism is also conserved. The chemical mechanism is often referred to as the two-metal-ion mechanism (Fig. 2) [24, 25] . Two divalent cations are required for polymerase-catalyzed nucleotidyl transfer; Mg 2+ is the biologically relevant cofactor. One metal ion (metal B) enters the active site in complex with the nucleotide substrate. The other metal ion (metal A) likely enters from solution after binding of the Mg 2+ -NTP complex. The metals are stabilized in the active site by coordination to conserved residues in motifs A and C, oxygens from all three phosphates of NTP, and the oxygen from the primer terminus. It is generally accepted that the role of metal A is to increase the nucleophilicity of the primer 3′-OH by lowering the pK a of the hydroxyl. Metals A and B interact with oxygens of the αphosphate so these metals could presumably increase the electrophilicity of the αphosphorous atom. Finally, metal B may also organize the triphosphate into a conformation Cameron et al.