Metal ions: supporting actors in the playbook of small ribozymes

Abstract

Since the 1980s, several small RNA motifs capable of chemical catalysis have been discovered. These small ribozymes, composed of between approximately 40 and 200 nucleotides, have been found to play vital roles in the replication of subviral and viral pathogens, as well as in gene regulation in prokaryotes, and have recently been discovered in noncoding eukaryotic RNAs. All of the known natural small ribozymes - the hairpin, hammerhead, hepatitis delta virus, Varkud satellite, and glmS ribozymes--catalyze the same self-cleavage reaction as RNase A, resulting in two products, one bearing a 2'-3' cyclic phosphate and the other a 5'-hydroxyl group. Although originally thought to be obligate metalloenzymes like the group I and II self-splicing introns, the small ribozymes are now known to support catalysis in a wide variety of cations that appear to be only indirectly involved in catalysis. Nevertheless, under physiologic conditions, metal ions are essential for the proper folding and function of the small ribozymes, the most effective of these being magnesium. Metal ions contribute to catalysis in the small ribozymes primarily by stabilizing the catalytically active conformation, but in some cases also by activating RNA functional groups for catalysis, directly participating in catalytic acid-base chemistry, and perhaps by neutralizing the developing negative charge of the transition state. Although interactions between the small ribozymes and cations are relatively nonspecific, ribozyme activity is quite sensitive to the types and concentrations of metal ions present in solution, suggesting a close evolutionary relationship between cellular metal ion homeostasis and cation requirements of catalytic RNAs, and perhaps RNA in general.

Figure 1. General mechanism of self-cleavage by the natural small ribozymes

(A) A Brønsted-Lowry base (β) abstracts a proton to activate the 2′-OH nucleophile, which then attacks the adjacent phosphate, forming a pentacoordinate transition state (B) with approximate collinearity between the 2′-oxygen, phosphorus atom, and 5′-oxygen leaving group – the in-line attack geometry. The negative charge of the transition state may be stabilized by one or several metal cations (Mⁿ⁺) that interact through inner- or outer-sphere contacts with the non-bridging oxygen atoms or by long-distance coulombic stabilization. A Brønsted-Lowry acid (α) donates a proton to the 5′-oxygen leaving group, resulting in a 5′-product bearing a 2′,3′-cyclic phosphate and a 3′-product bearing a 5′-OH (C).

Figure 2. Modes of metal ion binding to RNA

Metal cations (Mⁿ⁺) can associate with RNA via long-lived, specific interactions (A, B) requiring at least partial dehydration of the metal ion and RNA, or transient, diffuse interactions between the solvated RNA and metal ion (C). Specific interactions can involve direct chelation of the metal ion by RNA functional groups such as non-bridging phosphate oxygens (A), contacts mediated by inner-sphere water molecules (B), or a combination of the two.

Figure 3. Three-dimensional structures and metal ion binding sites of the natural small ribozymes

The ribozyme structures are shown in silver, divalent cations or probable binding sites in black, and the cleavage site in each ribozyme indicated by a black arrow. Crystal structures of (A) a hairpin ribozyme in the presence of Ca²⁺ ions [65], (B) a hammerhead ribozyme with Mn²⁺ ions [64], (C) the HDV ribozyme with Mg²⁺ ions [62], and (D) the glmS ribozyme in Mg²⁺ ions, with the necessary glucosamine-6-phosphate cofactor shown in dark gray [63]. Cocrystallized proteins and protein-binding domains of RNA used for crystallization purposes are not shown in these structures. E, Partial three-dimensional structure of the VS ribozyme derived from two similar low-resolution models [–143] (courtesy of Richard A. Collins and Ricardo Zamel), with black spheres indicating phosphates having probable direct contacts with divalent metal ions as revealed by phosphorothioate rescue with Mn²⁺ [72].