Structure-function relationships among RNA-dependent RNA polymerases

Abstract

RNA-dependent RNA polymerases (RdRPs) play key roles in viral transcription and genome replication, as well as epigenetic and post-transcriptional control of cellular gene expression. In this article, we review the crystallographic, biochemical, and molecular genetic data available for viral RdRPs that have led to a detailed description of substrate and cofactor binding, fidelity of nucleotide selection and incorporation, and catalysis. It is likely that the cellular RdRPs will share some of the basic structural and mechanistic principles gleaned from studies of viral RdRPs. Therefore, studies of the viral RdRP establish a framework for the study of cellular RdRPs, an important yet understudied class of nucleic acid polymerases.

Fig. 1

A-C Overall structures of RdRPs. Ribbon representations of RdRP structures (rainbow coloring with blue at the N-terminus and red at the C-terminus) bound to RNA template (black) and primer (gray) strands: A FMDV (1WNE) (Ferrer-Orta et al. 2004); B Bacteriophage ϕ6 (1HI0) (Butcher et al. 2001); C Reovirus (1N35) (Tao et al. 2002). Two views are presented for each structure, a “front” view down the axis of the RNA-binding, active site cleft (left panel) and a “side” or “back” view into the active site. Divalent metal ions at the active site in B and C are drawn as magenta spheres. Asp-338 in motif C of FMDV is drawn in space-filling representation as magenta spheres to mark the position of the active site in the absence of bound divalent metal ions

Fig. 2

A, B Structures of E·RNA·NTP complexes. A RNA primer-dependent elongation complex formed by reovirus RdRP (1N35) (Tao et al. 2002). B Primer-independent (de novo) initiation complex formed by bacteriophage ϕ6 RdRP (1HI0) (Butcher et al. 2001). Divalent metal ions are drawn as magenta spheres. Coordination and hydrogen bonds are drawn as dashed, red lines. The 3′-terminal residue of the RNA primer in reovirus RdRP is drawn in gray and the two residues of the RNA template that are complementary to the 3′-terminal residue of the RNA primer and the 3′-dNTP are drawn in black. The long, 4.5-Å distance between the 3′-OH of the primer and metal ion A is drawn in magenta as a dashed line

Fig. 3

Two-metal-ion mechanism for nucleotidyl transfer. The nucleoside triphosphate enters the active site with a divalent cation (Mg²⁺, metal B). This metal is coordinated by the β- and γ-phosphates of the nucleotide, by an Asp residue located in structural motif A of all polymerases, and likely water molecules (indicated as oxygen ligands to metal without specific designation). This metal orients the triphosphate in the active site and may contribute to charge neutralization during catalysis. Once the nucleotide is in place, the second divalent cation binds (Mg²⁺, metal A). Metal A is coordinated by the 3′-OH, the α-phosphate, and Asp residues of structural motifs A and C. This metal lowers the pK_a of the 3′-OH facilitating catalysis at physiological pH. (Adapted from Liu and Tsai 2001)

Fig. 4

Pentavalent phosphorane transition state. During the nucleotidyl transfer reaction, two proton transfer reactions must occur. The proton from the 3′-OH nucleophile must be removed; a proton must be donated to the pyrophosphate leaving group. To date there is no information on these steps of the nucleotidyl transfer reaction

Fig. 5

A, B De novo initiation and elongation complexes. A De novo initiation of RNA synthesis involves binding of the initiating nucleotide (GTPi; red) at the priming or initiation site (P-site;green box) and binding of the first NTP substrate (GTPi+1; blue) to the nucleotide binding site (N-site; white box). Specific binding sites for divalent cations (pink circles A and B) are shown in close proximity to the α-, β-, and γ-phosphates of the first nucleotide substrate. B Elongation complex. Nucleotide addition during elongation involves binding of the nascent RNA primer strand, positioning of the 3′-terminal nucleotide in the P-site, and binding of the first NTP substrate (i+1, blue) to the nucleotide binding site (N-site; white box)

Fig. 6

Elongation cycle. The stages of RNA synthesis can be divided into four steps: nucleotide binding (step 1), a conformational-change step, thought to be orientation of the triphosphate for catalysis (step 2), chemistry (step 3), and translocation (step 4)

Fig. 7

Structural basis for fidelity. The nucleotide-binding pocket of all nucleic acid polymerases with a canonical “palm”-based active site is highly conserved. The site can be divided into two parts: a region that has “universal” interactions mediated by conserved structural motif A that organize the metals and triphosphate for catalysis, and a region that has “adapted” interactions mediated by conserved structural motif B that dictate whether ribo- or 2′deoxyribonucleotides will be utilized. In the classical polymerase, there is a motif A residue located in the sugar-binding pocket capable of interacting with the motif B residue(s) involved in sugar selection. This motif A residue in other polymerases could represent the link between the nature of the bound nucleotide (correct vs incorrect) to the efficiency of nucleotidyl transfer as described herein for Asp-238 of 3D^pol. (Gohara et al. 2004)

Fig. 8

Alignment of conserved regions of RNAi RdRPs. Comparison of putative RdRP amino acid sequence from different organisms including tomato plant, Neurospora (QDE-1), C. elegans (EGO-1, RRF-1), Arabidopsis (SDE1), and Dictyostelium discoideum (RrpA). Amino acids in red indicate conserved residues in all sequences in the alignment. Those in blue and green indicate conservative substitutions and semi-conservative substitutions, respectively