Principles of ion recognition in RNA: insights from the group II intron structures

Abstract

Metal ions promote both RNA folding and catalysis, thus being essential in stabilizing the structure and determining the function of large RNA molecules, including group II introns. The latter are self-splicing metalloribozymes, containing a heteronuclear fourmetal-ion center within the active site. In addition to these catalytic ions, group II introns bind many other structural ions, including delocalized ions that bind the RNA diffusively and well-ordered ions that bind the RNA tightly with high occupancy. The latter ions, which can be studied by biophysical methods, have not yet been analyzed systematically. Here, we compare crystal structures of the group IIC intron from Oceanobacillus iheyensis and classify numerous site-bound ions, which are primarily localized in the intron core and near long-range tertiary contacts. Certain ion-binding sites resemble motifs observed in known RNA structures, while others are idiosyncratic to the group II intron. Particularly interesting are (1) ions proximal to the active site, which may participate in splicing together with the catalytic four-metal-ion center, (2) organic ions that bind regions predicted to interact with intron-encoded proteins, and (3) unusual monovalent ions bound to GU wobble pairs, GA mismatches, the S-turn, the tetraloop-receptor, and the T-loop. Our analysis extends the general principles by which ions participate in RNA structural organization and it will aid in the determination and interpretation of future RNA structures.

Structural Insights into the Mechanism of Group II Intron Splicing

“…These two Mg 2+ ions are located in positions supported by functional biochemical data on group II introns [22, 34, 35], and they have similar configurations as those observed in other ribozymes and protein enzymes that utilize two-metal-ion mechanisms for phosphodiester cleavage [36]. In addition, the two site-bound monovalent ions (K1 and K2, being either K + or NH4 + ) help to stabilize the proper conformation of the catalytic center (Figure 3B) [6, 30, 31]. …”

“…group II intron maintains a complex metal ion center at the catalytic core which includes two divalent ions (M1, M2), supported by two monovalent ions (K1, K2) (Figure 3B) [4, 6, 30, 31]. The two divalent ions (in most cases Mg 2+ ions) are located at a sharp kink formed by the 2-nt bulge in D5 (Figure 3B) with M1 and M2 coordinating the scissile phosphate, while M2 activating the nucleophile (Figure 3B) [6, 30, 31].…”

“…group II intron maintains a complex metal ion center at the catalytic core which includes two divalent ions (M1, M2), supported by two monovalent ions (K1, K2) (Figure 3B) [4, 6, 30, 31]. The two divalent ions (in most cases Mg 2+ ions) are located at a sharp kink formed by the 2-nt bulge in D5 (Figure 3B) with M1 and M2 coordinating the scissile phosphate, while M2 activating the nucleophile (Figure 3B) [6, 30, 31]. These two Mg 2+ ions are located in positions supported by functional biochemical data on group II introns [22, 34, 35], and they have similar configurations as those observed in other ribozymes and protein enzymes that utilize two-metal-ion mechanisms for phosphodiester cleavage [36].…”

“…For example, in the presence of Li + or Na + , which are too small to properly occupy the K1 and K2 sites, all 4 metal ions at the O.i. group II intron catalytic center are released from the catalytic core and rearrangements of active-site nucleotides are observed [6, 30, 31]. It will be important to determine how these metals are arranged when the active-site is occupied by the branch-site during each step of splicing.…”

Intron Biology, Focusing on Group II Introns, the Ancestors of Spliceosomal Introns

“…They form a structure consisting of a set of double helices resulting from Watson-Crick and Crick wobble base-pairing [ 12 ]. The six domains fold into a catalytically active tertiary structure with the assistance of a series of conserved motifs involved in long-range tertiary interactions surrounding a catalytic four-metal-ion center [ 13 ]. Some interactions involve Watson-Crick base pairs (α-α′, β-β′, γ-γ′, δ-δ′, ε-ε′, IBS1-EBS1, IBS2-EBS2 and IBS3-EBS3), whereas other are tetraloop-receptor interactions of known geometries (ζ-ζ′, η-η′ and θ-θ′), or other types of less well defi ned non-Watson-Crick interactions (λ-λ′, κ-κ′ and μ-μ′) [ 14 , 15 ].…”