Identification of catalytic metal ion ligands in ribozymes

Abstract

Site-bound metal ions participate in the catalytic mechanisms of many ribozymes. Understanding these mechanisms therefore requires knowledge of the specific ligands on both substrate and ribozyme that coordinate these catalytic metal ions. A number of different structural and biochemical strategies have been developed and refined for identifying metal ion binding sites within ribozymes, and for assessing the catalytic contributions of the metal ions bound at those sites. We review these approaches and provide examples of their application, focusing in particular on metal ion rescue experiments and their roles in the construction of the transition state models for the Tetrahymena group I and RNase P ribozymes.

Figure 1

Summary of techniques used to identify catalytic metal ion-ligand interactions within ribozymes.

Figure 2

A three-channel scheme describing the Mg²⁺-dependent and independent pathways for HDV ribozyme catalysis. In 1 M NaCl in the absence of Mg²⁺, the ribozyme reacts at a basal rate with rate constant k₁ (channel 1). Increasing concentrations of Mg²⁺ accelerate this rate in a log-linear fashion (channel 2) up to a point, after which the rate becomes independent of Mg²⁺ concentration (channel 3). The Mg²⁺ ion associated with channel 2 conditions is thought to be primarily structural, binding via inner sphere coordination to a base quadruple involving a protonated C41. Under channel 3 conditions, a second, catalytic, Mg²⁺ ion binds via outer sphere coordination in the vicinity of the active site. This fully hydrated Mg²⁺ ion is thought to participate in general acid-base catalysis. [Adapted from (10).]

Figure 3

EPR spectroscopy of a Mn²⁺ binding site within the hammerhead ribozyme. (A) EPR spectra of a 50 µM solution of Mn²⁺ alone (solid line) and in the presence of 10 µM of an RNA-DNA hybrid hammerhead ribozyme (dotted line). The addition of nucleic acid causes the six-line EPR spectrum of Mn²⁺ to diminish. (B) Model of the A9/G10.1 Mn²⁺ binding site in the tertiary-stabilized hammerhead ribozyme, as determined through energy minimization of crystallographic and ESEEM data. The Mn²⁺ ion coordinates the pro-R_P nonbridging oxygen of A9, the N7 of G10.1, and four water molecules. [Taken from (43) and (46), with permission.]

Figure 4

Raman spectroscopic investigation of HDV ribozyme crystals. (A) Raman difference spectra (pH 7.5 – pH 5.0) of crystals of the unmodified HDV ribozyme (top panel) and an HDV ribozyme in which 7-deazaguanosine has replaced the cleavage site G1 (bottom panel). The difference spectrum of the unmodified ribozyme shows peaks at 1489 and 323 cm⁻¹, thought to arise from metal ion coordination of a guanosine N7 and from a single inner sphere Mg²⁺ hydrate, respectively. Both features are lost when 7-deazaguanosine replaces guanosine at the cleavage site. (B) Stereo model of the HDV ribozyme active site, constructed by combining the Raman spectroscopic results with pre- and post-cleavage crystallographic data. The Mg²⁺ ion (magenta) directly coordinates the N7 of G1, along with five water molecules (grey spheres). [Taken from (13), with permission.]

Figure 5

Electrostatic potential surface of the P4–P6 domain from the Tetrahymena group I ribozyme, calculated using the NLPB equation. Regions of negative electrostatic potential appear red, while electropositive regions appear blue. The A-rich bulge that coordinates two Mg²⁺ ions is located in a pocket of highly negative electrostatic potential (“metal ion core”) with values ranging from −80 to −100 kT/e. [Adapted from (74), with permission.]

Figure 6

The Tetrahymena group I ribozyme. (A) Secondary structure. (B) Three-dimensional structure at 3.8 Å resolution (PDB file 1X8W). Mg²⁺ ions are represented as green spheres. (C) Transition state catalytic metal ion-ligand interactions within the Tetrahymena ribozyme active site, as deduced from metal ion rescue experiments.

Figure 7

Kinetic model of the Tetrahymena ribozyme reaction. The free enzyme (E) can bind the oligonucleotide substrate (S) to form the open complex, in the presence [(E·S·G)_O] or absence [(E·S)_O] of guanosine. The closed complex [(E·S·G)_C or (E·S)_C] forms when the P1 helix carrying the substrate “docks” into the active site of the ribozyme via the formation of tertiary interactions. [Taken from (111).]

Figure 8

Sequential discovery of three different catalytic metal ion-ligand interactions in the transition state of the Tetrahymena group I ribozyme reaction. (A) M_A coordinates the 3’ oxygen of the leaving group. (B) M_B coordinates the 3’-OH of the guanosine nucleophile. (C) M_C coordinates the 2’-OH of the guanosine nucleophile.

Figure 9

Mn²⁺ concentration dependence of the reaction of S_3’S relative to S_3’O (${\mathrm{k}}_{\text{obs}}^{\text{rel}}$) for the reaction shown in Scheme 1. The Mn²⁺-dependent acceleration of the reaction is associated with a specific affinity (K^Mn) of the rescuing Mn²⁺ ion for the free ribozyme. This affinity is a thermodynamic fingerprint for Mn²⁺ rescue of the 3’ sulfur modification. [Taken from (121).]

Figure 10

Both M_A and M_C coordinate the pro-S_P nonbridging oxygen of the scissile phosphate. (A) Model showing potential metal ion-ligand interactions involving the pro-S_P nonbridging oxygen (question marks). The grey sphere denotes a fourth divalent metal ion, a possibility not excluded from the results in (123). (B) Revised transition state model based on (124). Three metal ions are present in the active site, with metals A and C coordinating the pro-S_P nonbridging oxygen of the scissile phosphate.

Figure 11

Two Cd²⁺ ions at sites A and C rescue the reaction of S_3’S,P-S by the Tetrahymena ribozyme. The plot at left shows the Cd²⁺ concentration dependence of k^rel, the reaction rate constant for S_3’S,P-S relative to that of an all-oxygen substrate. The solid line fits the data according to a model in which two Cd²⁺ ions rescue the reaction, while the dashed line fits the data according to a model in which a single Cd²⁺ ion mediates rescue. [Data taken from (124).]

Figure 12

Mn²⁺ ions compete with Cd²⁺ ions for sites A and C during rescue of the S_3’S,P-S reaction. In the plot at left, the data are represented as filled spheres. The solid line shows the degree of competition expected from Mn²⁺ ions competing at sites A and C, while the dashed line shows the corresponding model for Mn²⁺ competition at sites A and B. [Data taken from (124).]

Figure 13

A single Cd²⁺ ion rescues the reaction in which G_2′NH2 replaces guanosine. The plot at left shows the Cd²⁺ dependence of the rate constant for the reaction involving G_2′NH2relative to guanosine for the all-oxygen substrate. The data are fit to a model in which a single Cd²⁺ ion rescues the reaction. [Data taken from (124).]

Figure 14

Two Cd²⁺ ions rescue the reaction of S_3’S,P-S with G_2′NH2. In the plot at left, the closed symbols denote two independent determinations of the Cd²⁺ dependence of the reaction of S_3’S,P-S with G_2′NH2, relative to that of the all-oxygen substrate with guanosine. The open symbols denote two independent determinations of k^rel for the reaction of S_3’S,P-S with guanosine, compared to that of the all-oxygen substrate with guanosine. The solid lines are fits of the data to a model in which two Cd²⁺ ions rescue the reaction, while the dashed lines are fits in which three Cd²⁺ ions rescue the reaction. [Data taken from (124).]

Figure 15

Sequential discovery of ligands within the Tetrahymena ribozyme that position the catalytic metal ions. (A) The pro-S_P nonbridging oxygen of C208 coordinates M_A. (B) The pro-S_P nonbridging oxygen of C262 coordinates M_C. (C) Additional ligands for M_A include The pro-S_P nonbridging oxygens of A304 and A306.

Figure 16

View of the active site of the Azoarcus group I ribozyme (PDB file 1ZZN). Nucleotide numbers in parentheses refer to homologous positions within the Tetrahymena ribozyme. Mg²⁺ ions are represented as green spheres. Black lines denote putative inner sphere interactions between ribozyme ligands and the metal ions.

Figure 17

View of the active site of the group I ribozyme derived from bacteriophage Twort (PDB file 1Y0Q). Numbering and colors are as in Figure 16.

Figure 18

Active site metal ion within the crystal structure of the Tetrahymena ribozyme (PDB file 1X8W). The guanosine at position 304 is one of several mutations introduced based on experiments that selected for thermostable variants of the ribozyme.

Figure 19

The RNA component of RNase P. (A) Secondary structure of E. coli P RNA. (B) Transition state model for the reaction catalyzed by RNase P. Question marks denote putative metal ion-ligand interactions awaiting experimental verification. [Adapted from (175), with permission.] (C) Crystal structure of the P RNA from B. stearothermophilus at 3.3 Å (PDB file 2A64). (D) Location (in red) of the P4 helix within the B. stearothermophilus P RNA.

Scheme 1