The contribution of metal ions to the structural stability of the large ribosomal subunit

Abstract

Both monovalent cations and magnesium ions are well known to be essential for the folding and stability of large RNA molecules that form complex and compact structures. In the atomic structure of the large ribosomal subunit from Haloarcula marismortui, we have identified 116 magnesium ions and 88 monovalent cations bound principally to rRNA. Although the rRNA structures to which these metal ions bind are highly idiosyncratic, a few common principles have emerged from the identities of the specific functional groups that coordinate them. The nonbridging oxygen of a phosphate group is the most common inner shell ligand of Mg++, and Mg++ ions having one or two such inner shell ligands are very common. Nonbridging phosphate oxygens and the heteroatoms of nucleotide bases are common outer shell ligands for Mg++ ions. Monovalent cations usually interact with nucleotide bases and protein groups, although some interactions with nonbridging phosphate oxygens are found. The most common monovalent cation binding site is the major groove side of G-U wobble pairs. Both divalent and monovalent cations stabilize the tertiary structure of 23S rRNA by mediating interactions between its structural domains. Bound metal ions are particularly abundant in the region surrounding the peptidyl transferase center, where stabilizing cationic tails of ribosomal proteins are notably absent. This may point to the importance of metal ions for the stabilization of specific RNA structures in the evolutionary period prior to the appearance of proteins, and hence many of these metal ion binding sites may be conserved across all phylogenetic kingdoms.

FIGURE 1.

(A) Experimental electron density map contoured at 0.8σ (blue mesh) showing typical electron density for a Mg²⁺ ion (gold). Water molecules of the inner-sphere octahedron are displayed as red spheres connected to a central Mg²⁺ ion with gold bonds. (B) Region of the experimental map (blue mesh) contoured at 0.8σ showing typical density for two Na⁺ ions (green). The corresponding region of an experimentally phased isomorphous difference map contoured at 4σ (red mesh) and calculated with coefficients corresponding to |F_obs(Rb+)| − |F_obs(native)| to a resolution of 3.7 Å is superimposed.

FIGURE 2.

Examples of each of six distinct geometric classes of Mg²⁺ ions observed in the large ribosomal subunit. The inner-sphere coordinations of each Mg²⁺ ion are represented with gold bonds. The outer-sphere coordinations of each Mg²⁺ ion are represented with thin black lines.

FIGURE 3.

Common binding sites for monovalent cations in the large ribosomal subunit. RNA is shown in a ball-and-stick representation with Na⁺ ions as green spheres. Thin black lines are shown between functional groups in the RNA that are observed to coordinate the Na⁺ ions.

FIGURE 4.

Structural comparison of A-platform binding sites for monovalent cations in the large ribosomal subunit (A) and the P4-P6 domain of the group I intron from Tertrahymena thermophila (B). Coordinates for (B) were taken from PDB #1GID. In both cases the AA-dinucleotide (red) is shown with surrounding RNA nucleotides (gray) and the monovalent cation (green).

FIGURE 5.

Structural comparison of two examples of a putative Mg²⁺ ion binding motif in 23S RNA. The G nucleotide (red) that coordinates the Mg²⁺ ion (gold) is shown in the context of surrounding nucleotides (gray).

FIGURE 6.

Magnesium ions that stabilize the tertiary and quaternary structures of the large ribosomal subunit. (A) A conserved interface between domains II (cyan), IV (green), and V (red) is shown with four Mg²⁺ ions (gold) that interact with the RNA backbone. All nucleotide bases are colored gray. (B) A Mg²⁺ ion (gold) that stabilizes an interface between domains II (cyan) and V (red), as well as ribosomal protein L3 (C atoms are gray, N atoms are blue, O atoms are red).

FIGURE 7.

Secondary structure diagram showing the metal ion binding sites present in the most conserved regions of 23S RNA. Nucleotides shown are 619–656, 749–906, 1299–1308, 1346–1374 in domain II, 1824–2025 in domain IV, and 2084–2127, 2266–2321, and 2419–2660 in domain V (H. marismortui numbering). Only nucleotides that coordinate the inner sphere of Mg²⁺ ions are shown connected to the ion.

FIGURE 8.

Three-dimensional representation showing the metal ion binding sites present in the peptidyl transferase center. The RNA backbone (red) is shown with bases (gray), Mg²⁺ ions (gold), and monovalent cations (green). Nucleotides shown are 2084–2127, 2266–2321, and 2419–2660 (H. marismortui numbering).