Abstract
The nucleotide incorporation fidelity of the viral RNA-dependent RNA polymerase (RdRp) is important for maintaining functional genetic information but, at the same time, is also important for generating sufficient genetic diversity to escape the bottlenecks of the host's antiviral response. We have previously shown that the structural dynamics of the motif D loop are closely related to nucleotide discrimination. Previous studies have also suggested that there is a reorientation of the triphosphate of the incoming nucleotide, which is essential before nucleophilic attack from the primer RNA 3'-hydroxyl. Here, we have used 31P NMR with poliovirus RdRp to show that the binding environment of the triphosphate is different when correct versus incorrect nucleotide binds. We also show that amino acid substitutions at residues known to interact with the triphosphate can alter the binding orientation/environment of the nucleotide, sometimes lead to protein conformational changes, and lead to substantial changes in RdRp fidelity. The analyses of other fidelity variants also show that changes in the triphosphate binding environment are not always accompanied by changes in the structural dynamics of the motif D loop or other regions known to be important for RdRp fidelity, including motif B. Altogether, our studies suggest that the conformational changes in motifs B and D, and the nucleoside triphosphate reorientation represent separable, "tunable" fidelity checkpoints.
Highlights
Genome maintenance and propagation are dependent on faithful and efficient nucleic acid replication catalyzed by a large superfamily of template-directed polymerases [1]
We have used NMR and kinetic studies to provide insight into the roles of motif F residues in the fidelity mechanism of PV RNA-dependent RNA polymerase (RdRp). 31P NMR studies have allowed us to probe the nucleoside triphosphate (NTP) triphosphate conformation for both cognate and noncognate NTP, and [methyl-13C]methionine NMR studies continue to provide insight into protein structural changes that accompany nucleotide selection, especially those associated with motifs B and D
31P NMR Spectra Used to Monitor NTP Conformations in PV RdRp—We propose that there are four events to NTP selection and incorporation in RdRps: initial Watson-Crick base pairing with the RNA template, interactions between the NTP ribose hydroxyls and residues in motifs A and B to induce further conformational changes, a realignment of the NTP triphosphate involving residues in motifs A and F, and conformational changes in motif D to bring in the general acid Lys359
Summary
RdRp, RNA-dependent RNA polymerase; PV, poliovirus; sym/sub-UA, symmetrical substrate RNA; MD, molecular dynamics; kpol, maximum polymerase rate constant; kpol/Kd(app), catalytic efficiency; SDKIE, solvent deuterium kinetic isotope effect; HSQC, heteronuclear single quantum coherence; AMP-CPP, adenosine 5Ј-(␣,-methylene)triphosphate; ␣-P and ␥-P, ␣- and ␥-phosphate, respectively; 6-FAM, 6-carboxyfluorescein; PDB, Protein Data Bank. Residues Lys-167 and Arg174 probably play important functional roles, including initial nucleotide binding, realignment of the triphosphate for nucleophilic attack, and stabilization of the negative charges in the pentacoordinate transition state [9, 16]. We have used NMR and kinetic studies to provide insight into the roles of motif F residues in the fidelity mechanism of PV RdRp. 31P NMR studies have allowed us to probe the NTP triphosphate conformation for both cognate and noncognate NTP, and [methyl-13C]methionine NMR studies continue to provide insight into protein structural changes that accompany nucleotide selection, especially those associated with motifs B and D. The results with the motif F and previously identified fidelity variants suggest that triphosphate alignment is an important fidelity checkpoint but may be independent of conformational changes that occur in motifs B and D
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