Figure 1.
(A) Cartoon representation of the QDE-1 ΔN P21 dimer–subunit A coloured according to domains: slab, blue; catalytic, purple; neck, pink; and head, orange; subunit B coloured grey. The approximate non-crystallographic 2-fold is represented as a grey line. Green spheres mark Mg2+ ions. Disordered regions correspond to ten residues at the N-terminus, 30 residues at the C-terminal, 45 residues in monomer A, and 48 in monomer B belonging to four loops (A: residues 590–603, 628–640, 1,241–1,251, and 1,271–1,281; B: 591–606, 627–640, 1,241–1,251, and 1,271–1,281).
(B) Topology of QDE-1 ΔN subunit A, coloured as in (A). The catalytic subdomains DPBB1, DPBB2, and flap are denoted by boxes. The non-crystallographic 2-fold is represented as in (A). Disordered loops are represented by dashed lines.
(C) View of presumed active site. The two DPBBs that form the active cleft, DPBB1 (residues 680–782) and DPBB2 (residues 916–1,018), are labelled. Sequence motifs conserved across other cRdRPs are highlighted: motif 1, red; 2, orange; 3, dark yellow, 4, purple; 5, dark pink; 6, bright pink; and 7, blue (see Figure S1). Invariant residues in cRdRPs are shown in ball-and-stick representation (O, red; and N, blue); Mg2+ ion shown as green sphere.
(D) Zoom of active site region, with conserved residues labelled. Representation is as for (C).
Table 1.
Refinement Statistics
Figure 2.
(A) Surface charge representation of subunit A of QDE-1 ΔN (subunit B coloured grey). Blue arrow indicates proposed RNA product groove; red arrow indicates proposed NTP tunnel; and purple arrow indicates tunnel linking active sites in the dimer. The Mg2+ ions are shown as green spheres. Left panel: dimer (view similar to Figure 1A). Right panel: sliced view of left panel to reveal the tunnels.
(B) Different conformations of QDE-1 ΔN subunits modify polymerase active site accessibility. Left panel: cartoon representation of superposition of observed subunit conformations, coloured according to domain definition: subunit A as in Figure 1A; subunit B: slab, cyan; catalytic, magenta; neck, wheat; and head, yellow; C2 crystal form subunit (semi-transparent): slab, marine; catalytic, violet; neck, light salmon; and head, orange. In all panels, Mg2+ ions are represented as green spheres. The direction of the movement of the slab and head domains is indicated by grey arrows, and the non-crystallographic 2-fold is shown. Central and right panels: RNA-DNA duplex (from an elongation complex of yeast RNApolII) fitted (using the operators for superposition of yeast RNApolII onto QDE-1) into QDE-1 ΔN closed and open subunits (represented as molecular surfaces). Centre panel colours subunit A (closed conformation). Right panel shows a view rotated by 180° with subunit B (open conformation) coloured. RNA-DNA duplex model is show in ball-and-stick representation.
(C) Close-up of the duplex model from (B). Domains are coloured as for the central panel of (B). The slab domain of molecule A would lie across the front of the figure and has therefore been removed for clarity.
Figure 3.
Stereo representation of the superposition of QDE-1 ΔN and yeast DdRP DPBBs (view as in Figure 1C). Structurally equivalent residues in QDE-1 ΔN are coloured dark purple (non-equivalent residues in light purple). Equivalent residues in yeast DPBBs are coloured green (non-equivalent residues in semi-transparent grey). QDE-1 ΔN and yeast (D481, D483, and D485) active site aspartates are coloured yellow and green respectively.
Figure 4.
Structure-Based Sequence Alignment of QDE-1 ΔN and DdRPs
Alignment of QDE1 ΔN (top sequence), bacterial (middle sequence, orange), and yeast (bottom sequence, green) polymerases is based on structurally equivalent residues, as determined by SHP [41]. Residues structurally equivalent in all polymerases are shaded purple, green (light for Rbp1, dark for Rp2) if equivalent in yeast DdRP and QDE1 ΔN, and orange if only equivalent in QDE1 ΔN and bacterial DdRP. Invariant residues are shaded in red. Conserved sequence motifs identified in cRdRPs are represented as in Figure S1, marked on QDE1 sequence. QDE1 secondary structure elements are shown on top, coloured according to domain definition (slab, blue; catalytic, deep purple; neck, pink; and head, red). DPBB1 and DPBB2 are outlined by deep purple boxes. The flap sub-domain and the potential “bridge helix” are also represented by boxes, coloured light purple and grey, respectively.
Figure 5.
(A) Evolutionary phylogenetic tree for known polymerases based on structural similarity (for description of the method see [43]). The “right-handed” polymerases, “double-barrel” polymerases, and polβ appear to form three separate, unrelated families. Within the “double-barrel” family, the DPBB2 domains containing the active site aspartate residues are more structurally conserved than DPBB1. Branches are coloured according to structural fold: green, right hand (dark, cellular; and light, viral), Polβ, blue; and DPBB-containing fold, dark magenta. Since we do not believe that all polymerases originate from a common ancestor, the central node of the tree is shaded grey. The key to the additional structures is: Yeast DPBB1, yeast RNApolII DPBB from Rbp2; Yeast DPBB2, yeast RNApolII DPBB from Rbp1; Bact. DPBB1, bacterial β subunit DPBB; Bact. DPBB2, bacterial β′ subunit DPBB; Polβ, rat DNA polymerase β; T7pol, bacteriophage T7 DdRP; KF, Klenow fragment of DNA polymerase I; HIV1-RT, human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT); λ3 reo, reovirus RdRP; Φ6, Φ6 bacteriophage RdRP; HCV, hepatitis C virus RdRP; BVDV, bovine viral diarrhoea virus RdRP; PV, poliovirus RdRP; RHDV, rabbit hemorrhagic disease virus RdRP; NV, Norwalk virus RdRP; HRV, human rhinovirus RdRP; and FMDV, foot–and-mouth disease virus RdRP.
(B) Originally, in an all RNA world, RNA self-replicates until the advent of a protein-based, primeval RNA-dependent RNA polymerase. Initially this possesses a single DPBB domain on a single polypeptide chain. Gene duplication leads to a polypeptide chain containing two copies of the DPBB domain. Differentiation of the two DPBB domains then results in QDE-1–like RdRPs. Emergence of DNA and associated increase of complexity lead to segregation of the DPBB into different polypeptidic chains, giving rise to the complex multi-subunit DdRP machinery observed today.