A recurrent magnesium-binding motif provides a framework for the ribosomal peptidyl transferase center.

The ribosome is an ancient macromolecular machine responsible for the synthesis of all proteins in all living organisms. Here we demonstrate that the ribosomal peptidyl transferase center (PTC) is supported by a framework of magnesium microclusters (Mg(2+)-muc's). Common features of Mg(2+)-muc's include two paired Mg(2+) ions that are chelated by a common bridging phosphate group in the form Mg((a))(2+)-(O1P-P-O2P)-Mg((b))(2+). This bridging phosphate is part of a 10-membered chelation ring in the form Mg((a))(2+)-(OP-P-O5'-C5'-C4'-C3'-O3'-P-OP)-Mg((a))(2+). The two phosphate groups of this 10-membered ring are contributed by adjacent residues along the RNA backbone. Both Mg(2+) ions are octahedrally coordinated, but are substantially dehydrated by interactions with additional RNA phosphate groups. The Mg(2+)-muc's in the LSU (large subunit) appear to be highly conserved over evolution, since they are unchanged in bacteria (Thermus thermophilus, PDB entry 2J01) and archaea (Haloarcula marismortui, PDB entry 1JJ2). The 2D elements of the 23S rRNA that are linked by Mg(2+)-muc's are conserved between the rRNAs of bacteria, archaea and eukarya and in mitochondrial rRNA, and in a proposed minimal 23S-rRNA. We observe Mg(2+)-muc's in other rRNAs including the bacterial 16S rRNA, and the P4-P6 domain of the tetrahymena Group I intron ribozyme. It appears that Mg(2+)-muc's are a primeval motif, with pivotal roles in RNA folding, function and evolution.

INTRODUCTION

Recent structures of large RNAs, such as the P4–P6 domain of the tetrahymena ribozyme (1–5), and larger RNAs such as ribosomes (6–14) continue to reveal important new principles of RNA structure and folding. Globular cores of large RNAs are characterized by base–base tertiary interactions and ‘buried’ phosphate groups (15,16). During RNA-folding cations are sequestered from bulk solvent, and held in close proximity to the polymer. Folding increases proximities of phosphate groups, and the electrostatic repulsion among them. Thus phosphate–phosphate repulsion must be offset by attraction between phosphates and cations.

Mg2+, since the beginning of life, has been closely associated with some of the central players in biological systems—phosphates and phosphate esters (17). Mg2+ shares a special geometric, electrostatic, thermodynamic relationship with phosphates (18). The ionic radius of Mg2+ is small (0.65 Å), the charge density is high, the coordination geometry is octahedral (the AOCN—average observed coordination number—is 5.98), the preferred ligands are charged or neutral oxygens, and the hydration enthalpy is large (−458 kcal/mol) (19–22). Ligand–ligand crowding is one of the hallmarks of Mg2+ complexes, leading to highly restrained ligand–Mg2+–ligand geometry, and strong ligand–ligand repulsive forces. In comparison with group I ions, Ca2+ or polyamines, Mg2+ has a much greater affinity for phosphate oxygens, and binds to them with well-defined geometry. Unlike other cations, Mg2+ brings phosphate oxygens in its first shell into direct contact with each other.

Chelation effects and topology influence interactions of nucleic acids with ions. For example magnesium forms a mononuclear motif with ADP/ATP in which one Mg2+ ion is chelated by a six-membered ring consisting of atoms Mg2+(a)–OαP-P-O-P-OβP–Mg2+(a) (18). Here we observe that a framework of dinuclear magnesium complexes Mg2+(a)–RNA–Mg2+(b) flanks the peptidyl transferase center (PTC) in LSU ribosomal structures (Figure 1). In these dinuclear clusters, two Mg2+ ions are chelated by a common bridging phosphate group. Additional structural features are conserved among these clusters, which are also identified in other RNAs, indicating that dinuclear RNA magnesium clusters compose by distinctive yet recurrent motif. This dinuclear Mg2+(a)–RNA–Mg2+(b) motif is referred to here as the magnesium microcluster (Mg2+-μc).

Figure 1.

Four Mg2+-μc's are observed in the LSU of H. marismortui (PDB entry 1JJ2). (A) View into the Peptidyl Transfer Center. The four Mg2+-μc's are represented as solid surfaces. The RNA atoms lining the polypeptide exit tunnel are accented in black. Mg2+-μc's D1, D2 and D4 encircle the PTC. Mg2+-μc's are colored: D1, purple; D2, yellow; D3, gray; D4, green. Ribosomal proteins and the 5S rRNA are omitted for clarity. (B) This view, looking across the polypeptide exit tunnel, is rotated by 90° relative to that of panel A. (C) The secondary structures of LSU rRNAs of H. marismortui [23S rRNA (7), dashed black line] and the mitochondrion of B. taurus [16S rRNA (51), red line]. Phosphate groups that are linked by magnesium ions within Mg2+-μc's are indicated by broad colored lines. The secondary structural elements that interact with Mg2+-μc's are conserved in these widely divergent LSUs. In the C. elegans LSU (not shown), the rRNA that binds to D3 is absent (52). The question mark indicates the portion of the mitochondrial B. Taurus LSU rRNA for which the secondary structure is unknown.

MATERIALS AND METHODS

Molecular interactions

Ribosomal and other RNA structures were obtained from the PDB (23) and subject to automated (24–28) and manual structural analysis and decomposition. Because its resolution is greatest, the Haloarcula marismortui LSU is used as the benchmark and the basis for comparison. First, shell Mg2+–ligand interactions are defined by distances less than 2.4 Å (18). The Mg2+ positions in 23S-rRNAHM are, as determined by various geometric criteria, highly credible, with a few exceptions. The Mg2+ to oxygen distance is an excellent metric in that the predicted and observed (in 23S-rRNAHM) frequencies reach distinct maxima at 2.1 Å and fall to nearly zero by 2.6 Å. The thermal factors of the Mg2+ atoms in the Mg clusters do not suggest partial occupancy (i.e. they are not in general significantly higher than nearby atoms.) As indicated by relative OP (phosphate oxygen atom) positions, essentially all relevant Mg2+ ions were added correctly to the 1JJ2 model. An exhaustive survey of all Mg2+–RNA interactions in the database was conducted with the MeRNA database (29). Hydrogen bond distances are defined by distances less than 3.4 Å between hydrogen bond donating and accepting heavy atoms.

Sequence homology and alignment

The fragment of ribosomal protein L2 that associates with the H. marismortui D2 complex (PNVRGVAMNAVDHPFGGG, peptide L2/D2) was determined by inspection of the 3D structure (1JJ2). That sequence was used as a search string in Blast (30), finding matches in all cytoplasmic and chloroplast ribosomes and in fungal mitoribosomes, but not in other mitoribosomes or in other chloroplast ribosomes. Exhaustive searching failed to find L2/D2 homologs in nonfungal mitoribosomes. Protein alignment was performed with Clustalw (31).

Structural alignment

To date, structural alignment of very large RNAs remains challenge due to the large size, intricate backbone choreography and tertiary interactions. We developed a heuristic method, using a ‘Divide and Conquer’ strategy for performing whole body superimposition of 23s rRNAs. With this method (to be described elsewhere), the alignment and superimposition of the 23s rRNAs of Thermus thermophilus and H. marismortui gives an overall RMSD of atomic positions of 1.2 Å. This superimposition utilizes 73% of RNA backbone atoms (around 2129 residues). This accurate superimposition allows one to identify conserved Mg2+ ions.

RESULTS

Forty-one Mg2+ ions in H. marismortui LSU were determined by geometric analysis to be highly coordinated by rRNA phosphate groups [more than one phosphate group in the Mg2+ first shell, also see (11)]. Visual inspection of each of these Mg2+ ions revealed that four pairs are distinctive environments, leading to our classification of them as Mg2+-μc's.

The basic motif of the Mg2+-μc is defined by several features (Figure 2A). Two paired Mg2+ ions are chelated by a common bridging phosphate group in the form Mg2+(a)–(O1P-P-O2P)–Mg2+(b). This bridging phosphate is part of a 10-membered chelation ring in the form Mg2+(a)–(OP-P-O5′-C5′-C4′-C3′-O3′-P-OP)–Mg2+(a). The two phosphate groups of this 10-membered ring are contributed by adjacent residues along the RNA backbone. Both Mg2+ ions are octahedrally coordinated, but are substantially dehydrated by interactions with additional RNA phosphate groups.

Figure 2.

The Mg2+-μc motif. (A) A schematic diagram illustrating the Mg2+(a)–(O1P-P-O2P)–Mg2+(b) bridge (outlined in blue), the 10-membered chelation ring (yellow), an unstacked base and the additional RNA phosphate groups that enter the Mg2+ first shell at variable positions. Carbon is green, oxygen is red and phosphorous is orange. Magnesium (a) is cyan while magnesium (b) is brown. (B) Superimposition of four Mg2+-μc's (D1 purple, D2 yellow, D3 gray and D4 green) from the H. marismortui LSU. All of the atoms shown here were used in the superimposition of the clusters except for the RNA bases.

The conformation of the RNA and the positions of the Mg2+ ions are substantially conserved in a Mg2+-μc. The position and conformation of the RNA is constrained by the Mg2+ ions. The relative positions of the Mg2+ ions are constrained by the Mg2+(a)–(O1P-P-O2P)–Mg2+(b) bridges. The superimposition of four Mg2+-μc's is shown in Figure 2B. The RMSD of the atomic positions of this superimposition is 0.59 Å.

Although the basic motif is conserved in all four clusters, Mg2+-μc's are elaborated by additional RNA ligands. RNA phosphate groups that are not part of the bridging phosphate or the 10-membered ring can enter the Mg2+ coordination sphere at various positions. Each Mg2+-μc is defined in toto by two paired Mg2+ ions plus all RNA residues that engage in first shell interactions with the paired Mg2+ ions. Therefore each Mg2+-μc has a unique shape. Specific descriptions of the four clusters are provided here.

Mg2+-μc D2

In this cluster, the Mg2+(a)–(O1P-P-O2P)–Mg2+(b) bridge is composed of the phosphate group of G877 along with Mg2+ ions 8003 and 8013 (Figure 3A). The 10-membered Mg2+(a)–(OP-P-O5′-C5′-C4′-C3′-O3′-P-OP)–Mg2+(a) system is composed of the phosphate group and ribose atoms of A876 along with the phosphate group of G877. Both phosphates from that segment of RNA chelate Mg2+ 8003.

Figure 3.

Atomic level representations of the four Mg2+-μc's of H. marismortu LSU (PDB entry 1JJ2). The bridging phosphates group are outlined in blue and the 10-membered chelation rings are shaded in yellow. (A) Top: Mg2+-μc D2 with bases, riboses (except C3′, C4′, C5′) and protein sidechains omitted (Mg2+ ions 8003 and 8013). A three residue fragment of ribosomal protein L2 contains universally conserved asparagine (i) and methione (h). Bottom: Mg2+-μc D2 with bases, riboses and protein sidechains included. (B) Top: Mg2+-μc D4 with bases and riboses omitted (Mg2+ ions 8005 and 8007). This Mg2+-μc has two 10-membered chelation rings. Bottom: Bases and riboses are included. (C) Top: Bases and riboses omitted (Mg2+ ions 8001 and 8002). This Mg2+-μc has two Mg2+(a)-O1P-P-O2P-Mg2+(b) bridges (phosphates of residues C2534 and A2483). Bottom: Bases and riboses are included. (D) Top: Mg2+-μc D3 with bases and riboses omitted (Mg2+ ions 8016 and 8029). Bottom: Bases and riboses are included. First-shell Mg2+ contacts are black solid lines. Hydrogen bonds are dashed lines. Carbon is green, oxygen is red, phosphorous is orange and magnesium is yellow. Residue labels in parentheses are from T. Thermophilus, using the E. coli numbering scheme.

The two Mg2+ ions 8003 and 8013 of Mg2+-μc D2 jointly link RNA segments that are remote in the linear sequence and in the secondary structure (32). These linkages take the form of first shell OP(Mg2+-OP(), where j >> i. Mg2+ 8003 links the phosphates of A876 and G877 to the phosphate of A2624. Mg2+ 8013 links the phosphate of G877 to the phosphate of G2623. The Mg2+-μc D2 linkages are indicated by the long yellow strips in the secondary structure in Figure 1C.

The first shell RNA–Mg2+ interactions unstack the bases (Figure 3A, bottom panel). Residue A876 is unstacked from G877, with a C1′–C1′ distance (A876 to G877) of 7.2 Å. Residue A2622 is unstacked from G2623 (rC1′–C1′ = 7.4 Å). This unstacking is important for assembly. The unstacked face of G877 forms part of the cavity for protein L2 (below). A2622 (unstacked in Mg2+-μc D2) intercalates between U1838 and A1839 (Mg2+-μc D4, below).

Mg2+-μc D4

In this cluster the Mg2+(a)–(O1P-P-O2P)–Mg2+(b) bridge is composed of the phosphate group of U1839 along with Mg2+ ions 8005 and 8007 (Figure 3B). There are two 10-membered ring systems. One Mg2+(a)–(OP-P-O5′-C5′-C4′-C3′-O3′-P-OP)–Mg2+(a) system is composed of the phosphate group and ribose atoms of U1838 plus the phosphate group of A1839, along with Mg2+ 8005, which is also chelated by the phosphate of A1836. A second Mg2+(a)–(OP-P-O5′-C5′-C4′-C3′-O3′-P-OP)–Mg2+(a) system is composed of the phosphate group and ribose atoms of A1839 along plus the phosphate group of A1840. This RNA segment chelates Mg2+ 8007.

These Mg2+–RNA interactions induce an extended array of unstacked bases (Figure 3B, bottom panel). Residue U1835 is unstacked from A1836 (rC1′–C1′ = 9.6 Å). Residue A1836 is unstacked from G1837 (rC1′–C1′ = 6.6 Å). Residue G1837 is unstacked from U1838 (rC1′–C1′ = 8.8 Å). Residue U1838 is unstacked from A1839 (rC1′–C1′ = 7.7). Residue A1839 is unstacked from A1840 (rC1′–C1′ = 7.8 Å). These unstacked bases of Mg2+-μc D4 provide a complementary docking surface for Mg2+-μc D2 (Figure 4). As noted above A2622 (of Mg2+-μc D2) intercalates between U1838 and A1839 (of Mg2+-μc D4).

Figure 4.

The complex formed by Mg2+-μc's D4 and D2 and the loop-L2/D2 of ribosomal protein L2. Structures of both H. marismortui and T. Thermophilus are shown. Magnesium ions are indicated by spheres. When the 23S rRNAs of H. marismortui and T. thermophilus are superimposed using 73% of rRNA backbone atoms, the RMSD of atomic positions of the eight Mg2+ ions within the four Mg2+-μc's is very small, only 0.4 Å.

Mg2+ ion 8007 of Mg2+-μc D4 links RNA segments that are remote in the linear sequence and in the secondary structure. This Mg2+ ion links the phosphates of A1839 and A1840 and to the phosphate of U832. This linkage is indicated by the long green strip in the secondary structure in Figure 1C.

Mg2+-μc D1

This cluster has a double bridge. One Mg2+(a)–(O1P-P-O2P)–Mg2+(b) bridge is composed of the phosphate group of C2534 along with Mg2+ ions 8001 and 8002 (Figure 3C). A second Mg2+(a)–(O1P-P-O2P)–Mg2+(b) linkage is composed of the phosphate group of A2483, which joins the same two Mg2+ ions. The double bridge is associated with a decreased Mg2+–Mg2+ distance, as illustrated by the shift of one Mg2+ relative to its homologs in Figure 2B. The 10-membered Mg2+(a)–(OP-P-O5′-C5′-C4′-C3′-O3′-P-OP)–Mg2+(a) system is composed of the phosphate group and ribose atoms of C2533 along with the phosphate group of C2534. This segment of RNA chelates Mg2+ 8001.

Both Mg2+ ions link RNA segments that are not adjacent in the linear sequence or secondary structure. The OPs of A2483 are linked to the phosphates of C2533 (one OP) and C2534 (both OPs) in a joint interaction involving both Mg2+ ions. These RNA–Mg2+ interactions unstack G2482 from A2483 (Figure 3C, bottom panel; rC1′–C1′ = 8.3 Å). In addition, A2532 is unstacked from C2533 (rC1′–C1′ = 6.9 Å).

Mg2+-μc D3

The Mg2+(a)–(O1P-P-O2P)–Mg2+(b) bridge in this cluster is composed of the phosphate group of C1679 along with Mg2+ ions 8016 and 8029 (Figure 3D). The 10-membered Mg2+(a)–(OP-P-O5′-C5′-C4′-C3′-O3′-P-OP)–Mg2+(a) system is composed of the phosphate group and ribose atoms of A1678 along with the phosphate group of C1679. This segment of RNA chelates Mg2+ 8016.

Both Mg2+ ions link RNA segments that are not adjacent in the linear sequence or secondary structure. An OP of A1678 and an OP of C1679 are linked by Mg2+ 8016 to an OP of A1504. An OP of C1679 is linked via Mg2+ 8029 to an OP of U1503. These RNA–Mg2+ interactions unstack the bases (Figure 3D, bottom panel). U1677 is unstacked from A 1678 (rC1′–C1′ = 8.0 Å). Residue A1502 is unstacked from U1503 (rC1′–C1′ = 7.7)

Role of Mg2+-μc's in activity

Three Mg2+-μc's (D1, D2 and D4) flank the PTC. These Mg2+-μc's are not directly involved in catalysis, and do not form the innermost layer of the PTC, but provide the framework and supporting structure for RNA that does. Mg2+-μc's lend critical support to function by forming convoluted binding surfaces and providing rigid frameworks for attachment and buttressing of catalytic residues.

The fourth cluster (D3) is located near the exit site of the polypeptide exit tunnel. Previously Steitz and Moore (11,32) showed that within the LSU, the concentration of Mg2+ ions is greatest near PTC. They also described one ‘magnesium cluster’ (part of Mg2+-μc D2).

Mg2+-μc—ribosomal protein interactions

Mg2+-μc D2 is associated with ribosomal protein L2. Inspection of the structures reveal an 18 amino-acid loop of L2 (loop-L2/D2, Table 1) forms a binding pocket for Mg2+-μc D2. As shown in Figures 3A and 4, methionine (h) and valine (c) along with the backbone carbonyl of an alanine (g) form a cleft for A875 (A782TT). Asparagine (i) and the carbonyl oxygen of alanine (g) bind to the first shell water molecules of the Mg2+ ions. Histidine (m) forms part of the tightly packed core of loop-L2/D2 (data not shown). The conformation of loop L2/D2 is highly conserved between T. thermophilus and H. marismortui (Figure 4). The RMSD of atomic positions of loop-L2/D2 is 0.6 Å in the globally superimposed LSUs (H. marismortui versus T. thermophilus, using all atoms of 18 residues except for differing sidechains for the RMSD calculation, 110 atoms total).

Table 1.

Mg2+-μc D2-binding loop of ribosomal protein L2 (loop-L2/D2)

aThe eukaryotic equivalent of ribosomal protein L2 is L8. The mitochondrial equivalent of L2 is rml2. Blue text indicates conserved sequences in all ribosomes including mitoribosomes. Red text indicates conserved sequences in all cytoplasmic and chloroplast ribosomes and in fungal mitoribosomes, but not in other mitoribosomes. The ordering of this table was obtained from the complete L2/L8/rml2 sequence alignment with ClustalW (31).

bLoop-L2/D2 is bold. Observed sequence changes of loop-L2/D2 are conservative.

cPositions of loop-L2/D2 are defined by amino acid positions a–r.

dLoop-L2/D2 contains ribosomal protein L2 residues 187–204 in H. marismortui.

eLoop-L2/D2 contains ribosomal protein L2 residues 219–226 in T. thermophilus.

fLoop-L2/D2 contains ribosomal protein rml2 residues 331–348 in S. cerevisiae. Mitochondrial and chloroplast ribosomes are thought to have undergone major remodeling (51,53,54) and are the most divergent from other ribosomes.

gThe H. sapiens mitoribosome lacks loop-L2/D2 as do other non-fungal mitoribosomes.

Mg2+-μc's in other RNAs

As shown in the Supplementary Data, Mg2+-μc's are observed not only in the 23S rRNA, but also in the bacterial 16S rRNA (8), the P4–P6 domain of the tetrahymena Group I intron ribozyme (2). A related magnesium cluster is found in Group II intron ribozyme (33).

DISCUSSION

Mg2+-μc structure

Four Mg2+-μc's (Microclusters D1, D2, D3 and D4; Figure 1) are found within the LSU of H. marismortui [an archaebacterium, PDB entry 1JJ2 (7)]. These four Mg2+-μc's are also found within the LSU of T. thermophilus [a bacterium, PDB entry 2J01 (13)]. The Mg2+-μc's are highly conserved in position, conformation and interactions between the two LSUs. Mg2+-μc's are defined by common features (Figure 2A) including (i) chelation of two Mg2+ ions by a common bridging phosphate in the form of Mg2+(a)–(O1P-P-O2P)–Mg2+(b), (ii) chelation of one of these Mg2+ ions by phosphate groups of adjacent residues in the form of Mg2+(a)–(OP-P-O5′-C5′-C4′-C3′-O3′-P-OP)–Mg2+(a), (iii) unpaired and unstacked bases with non-canonical RNA conformations and (iv) and close proximity to regions of function.

Mg2+-μc's are unique structural entities with rigidity and forced dispositions of functional groups that would be difficult to achieve by RNA alone or by RNA in association with group 1 cations. Mg2+-μc's by nature of their Mg2+(a)–(O1P-P-O2P)–Mg2+(b) linkages impose unusual constraints on RNA conformation and force unstacking of bases. The bridging phosphate is, like all phosphates, restricted to tetrahedral geometry. The ligands of Mg2+ ions are restricted to octahedral geometry. Mg2+-μc is composed of two octahedra linked by a tetrahedral phosphorous atom. Therefore, the core of each cluster is geometrically rigid and tightly packed. In addition, it can be seen that the RNA of Mg2+-μc's form intricate and convoluted surfaces (Figure 4), in part facilitating their association with other RNA and with protein.

Ordinarily, the conformational space accessible to RNA is rather limited, and is driven by stacking interactions and double-strand formation. Noller (34) has suggested that small molecules can extend the repertoire of RNA structure, and may have performed that role during early evolution. Magnesium, like a small molecule, can drive RNA into unusual conformation states (11,18). Mg2+-μc's demonstrate not only Mg2+-driven deviation from canonical stacked conformations, but show how these altered states increase surface undulation, and facilitate highly specific interactions, such as those between Mg2+-μc D2 and Mg2+-μc D4 and those between Mg2+-μc D2 and loop-L2/D2 of ribosomal protein L2 (Figure 4).

RNA–protein interactions

Mg2+-μc D2 is associated with ribosomal protein L2 in the LSU. Mutations in ribosomal protein L2 degrade ribosome activity (35–38). The amino-acid sequence of the N-terminal region of ribosomal protein L2 appears to be among the most highly conserved in the phylogenic tree (Table 1). An 18 amino-acid loop of L2 (loop-L2/D2) forms a binding pocket for Mg2+-μc D2. Ten amino-acid residues of loop-L2/D2 are universally conserved in all cytoplasmic and chloroplast ribosomes and in fungal mitoribosomes. Mutations of other residues of loop-L2/D2 are infrequent and are between analogous amino acids such as aspartic acid and glutamic acid or between valine and threonine.

Prior to the availability of ribosome structures in 2000, a direct role for rL2 in catalysis appeared to be consistent with phylogenic, mutagenesis and biochemical data. But a catalytic role for L2 is ruled out by the realization that the ribosome is a ribozyme (10,39,40). The strict sequence conservation of loop-L2/D2 appears to arise from a requirement for complementarily of the L2 protein surface with that of Mg2+-μc D2 (Figures 3A and 4). Because its mutation knocks out PTC activity, His(m) (i.e. histidine residue m of loop L2/D2, Table 1) was previously thought to be part of a serine-protease like charge-relay system. It now appears that the importance of His(m) arises from stabilization of loop-L2/D2 in the appropriate conformation for association with Mg2+-μc D2. The conformation of loop L2/D2 is highly conserved between T. thermophilus and H. marismortui (Figure 4).

Microclusters in other RNAs

Mg2+-μc's are observed in other ribozymes. A Mg2+-μc, shown in the Supplementary Data, is observed in the P4–P6 domain of the tetrahymena Group I intron ribozyme (2). A Mg2+-μc in the 16S rRNA of T. thermophilus [PDB entry 1FJG (41)], appears to be disrupted upon ribosomal assembly [PDB entry 2J00 (13)]. The SARS s2m RNA described by Scott contains a pair of Mg2+ ions linked by a single phosphate (42), but those Mg2+ are otherwise fully hydrated, with no additional RNA ligands and so do not constitute a Mg2+-μc.

Reasonable coordination geometry in a Mg2+-μc results in Mg2+-Mg2+ distances of 5.3–5.6 Å (except for the doubly bridged D1 cluster, with a Mg2+–Mg2+ distance of 4.7 Å). Mg2+-μc's differ from the Mg2+ complexes proposed in the two-Mg2+ catalyzed phosphoryl-transfer mechanism (43,44). In those complexes, a phosphate oxygen (not a phosphate group) bridges two Mg2+ ions, with a Mg2+–Mg2+ distance of 3.9 Å.

Evolution

Our discussion here makes use of the basic elements of the comparative approach (45) where history is reconstructed by degree of similarity between homologous structures or sequences. Biologists have long used macroscopic structure (skeletal, cellular, etc.) to infer phylogenetic relationships. The logic of that approach extends to the level of macromolecular structure. Comparison between homologous macromolecular structures can provide information on very distant evolutionary events, because macromolecular structure changes more slowly than sequence over evolutionary time (46,47).

The Mg2+-μc's in the LSU appear to be highly conserved over evolution, since they are unchanged in bacteria (T. thermophilus) and archaea (H. marismortui), which are thought to have diverged at the LUCA, several billions of years ago (48). Mg2+-μc's are found in association with the most conserved rRNA secondary structures (Figure 1C). Mg2+-μc's link these conserved secondary structural elements via ‘electrostatic tertiary interactions’, which are composed of phosphate-Mg2+-phosphate interactions. As shown in Figure 1C, the 2D elements of the 23S rRNA that are linked by Mg2+-μc's are conserved between the rRNAs of bacteria, archaea and eukarya (49) and the LSU rRNA of mitochondria (50,51), and in a proposed minimal LSU rRNA (52). The exception is Mg2+-μc D3, which has been dispensed of in some mitoribosomes (such as that of Caenorhabditis elegans) by conversion of the RNA-based polypeptide exit tunnel to a protein-based tunnel (51).

We suggest that Mg2+-μc's represent a primeval motif, with roles in RNA folding and catalysis that have found utility over deep evolutionary history. The Mg2+-μc's are located at functionally important regions of the ribosome, and are associated with unusual conformational states of RNA, forming intricate binding surfaces.

Mg2+-μc's D1, D2 and D4 but not loop-L2/D2 are conserved in all mitoribosomes. Mitoribosomes have been substantially remodeled over time, as can be seen by comparison of extant mitoribosomes with those of the ancestral endosymbiont (51,53,54). Mitoribosomes have twice the protein and half the rRNA of the bacterial ribosome. Although rRNA secondary elements that contain Mg2+-μc's D1, D2 and D4 are conserved all mitoribosomes, loop-L2/D2 appears to be absent from mitoribosomes other than those of fungi (Table 1). In the human mitoribosome protein L8 (the mammalian equivalent of L2), the N-terminus has been replaced by a sequence that diverges widely from loop-L2/D2 (Table 1). It may be that loop-L2/D2 has been structurally replaced by a nuclear encoded protein with unrelated sequence.

SUMMARY

The ribosomal peptidyl transferase center (PTC) is supported by a framework of magnesium microclusters (Mg2+-μc's). Mg2+-μc's are characterized by direct phosphate chelation of two magnesium ions in the form of Mg2+(a)–(O1P-P-O2P)–Mg2+(b), phosphate groups of adjacent RNA residues as first-shell ligands of a common Mg2+ ion, and undulated RNA surfaces with unpaired and unstacked bases.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

FUNDING

This work was funded in part by the NASA Astrobiology Institute. Funding for open access charge: NASA Astrobiology Institute.

Conflict of interest statement. None declared.