Crystal structure of the catalytic core of an RNA-polymerase ribozyme

Abstract

Primordial organisms of the putative RNA world would have required polymerase ribozymes able to replicate RNA. Known ribozymes with polymerase activity best approximating that needed for RNA replication contain at their catalytic core the class I RNA ligase, an artificial ribozyme with a catalytic rate among the fastest of known ribozymes. Here we present the 3.0 angstrom crystal structure of this ligase. The architecture resembles a tripod, its three legs converging near the ligation junction. Interacting with this tripod scaffold through a series of 10 minor-groove interactions (including two A-minor triads) is the unpaired segment that contributes to and organizes the active site. A cytosine nucleobase and two backbone phosphates abut the ligation junction; their location suggests a model for catalysis resembling that of proteinaceous polymerases.

Fig. 1

Global architecture of the ligase ribozyme. (A) Secondary structure and reaction scheme of a ligase variant with decreased Mg²⁺ dependence (10). It is depicted undergoing ligation, using the classical secondary-structure representation (15). Red arrows indicate attack by the substrate 3′-hydroxyl on the ribozyme α-phosphate with concomitant loss of pyrophosphate. (B) Revised secondary structure of the crystallization construct, reflecting the coaxial stacking and relative domain orientation. Indicated is the ligation junction (thick red dash), backbone phosphates at the active site (yellow dashes), base triples (boxed residues connected with gray lines), and stacking interactions (residues vertically aligned or connected with gray lines terminating in gray bars). Residues numbered as in (A); those in gray were added to facilitate crystallization. Base-pair geometries indicated using nomenclature of (27). (C) Ribbon representation of ligase structure, as if peering into the active site (yellow) and ligation junction (red). (D) Top-down view, rotated ~90° relative to (C).

Fig. 2

Tertiary contacts involving the three longest joining regions. (A) Interactions bridging the three domains. (B) The path of J1/3. (C) Hydrogen bonds of the two A-minor triads (fig. S8).

Fig. 3

Architecture of the active site. (A) The active site, as viewed from the ligation junction, removing P1–P2 for clarity. (B) Interactions near G1:C12, which is analogous to the NTPtemplate pair during polymerization (2-4, 9). Meshes are simulated-annealing OMIT maps in which active-site nucleotides (gray, contoured at 2σ) or the hydrated metal cluster (aqua, 4σ) were excluded from map calculations. (C) Stereograph of the active site. Black dashes indicate hydrogen bonds; magenta dashes indicate proximity between A29 and C30 phosphate oxygens and the ligation junction (red). Mesh represents a simulated-annealing OMIT map (4.5 σ) in which the hydrated metal was excluded from map calculations. (D) Mean interference values (±SD) from three α-phosphorothiolate NAIM experiments. The secondary structure is aligned above. Strong interference was truncated at the detection limit, 6.0 (10, 22). Missing positions are those modified to facilitate crystallization (hashes) or too close to the termini to measure.

Fig. 4

Transition-state stabilization by polymerases built from either protein or RNA. (A) Catalysis by proteinaceous polymerases (23, 24). Indicated are bonds formed or broken during the transition state (red arrows), coordination of catalytic metal ions, M_A and M_B (gray solid lines), and an active-site acid (A•••H). (B) Model for catalysis by the ligase ribozyme. Notation as in (A), with the addition of a hydrogen bond between C47 N4 and the leaving group (dashed gray line). Some magnesium ligands are not specified; for those that are, relative orientations are unknown. A proposed contact to the reactive phosphate pro-R_P oxygen (28) and two speculative contacts implied by NAIM are in blue. Features not supported (or refuted) by structural or biochemical evidence are in gray.