A glimpse into the active site of a group II intron and maybe the spliceosome, too

Abstract

The X-ray crystal structure of an excised group II self-splicing intron was recently solved by the Pyle group. Here we review some of the notable features of this structure and what they may tell us about the catalytic active site of the group II ribozyme and potentially the spliceosome. The new structure validates the central role of domain V in both the structure and catalytic function of the ribozyme and resolves several outstanding puzzles raised by previous biochemical, genetic and structural studies. While lacking both exons as well as the cleavage sites and nucleophiles, the structure reveals how a network of tertiary interactions can position two divalent metal ions in a configuration that is ideal for catalysis.

FIGURE 1.

Parallels between the splicing mechanisms of the spliceosome and group II introns. (Left panel) spliceosomal pre-mRNA splicing. Group II intron splicing using the branching (middle panel) and hydrolytic pathways (right panel) are depicted, together with the diastereomeric preferences (Rp or Sp) for each step of the splicing reactions. A conformational rearrangement is required between catalytic steps 1 and 2.

FIGURE 2.

Domain V is at the center of the group II intron structure. (A) Revised secondary structure of the group II intron based on the crystal structure of the O. iheyensis intron. The breakdown of the domain structure reflecting Watson–Crick tertiary interactions and coaxial stacking is depicted and the 5′ and 3′ ends of the intron are circled. The active site centered on domain V is depicted in the shaded box. (B) The region centered on domain V is expanded to depict details of long-range interactions involving conserved regions of domain V and the other intronic elements. These include the internal bulge adenosine in domain V that forms a Φ interaction with ε element in domain 1—I(i); the tetraloop within domain V that docks into the TLR via the ζ–ζ′ interaction; the upper helix of domain V that interacts with the IC element in domain I via the λ–λ′ interaction; and the lower helix of domain V that interacts with the ID1 element in domain I via the K–K′ interaction. The η–η′ interaction shown here between domains II and VI is absent in the crystallized construct. (C) Domains I, II, III, and IV, shown here as colored surfaces corresponding to the colors in A with the structures outlined as ribbon, completely encase domain V. Domain V is shown as a red cartoon with Watson–Crick pairings outlined. The J2/3 element is shown in purple and the EBS1 element in cyan. The catalytically implicated Mg²⁺ ions are presented as gray spheres and a third Mg²⁺ ion that binds to the tetraloop is depicted as a black sphere. The K⁺ ions are omitted for clarity.

FIGURE 3.

Details of the catalytic triplex structure. The conserved linker element J2/3 (blue) forms a base triple interaction with catalytic triad elements (5′-triad nucleotides in violet and the 3′-triad nucleotides in orange) and stacks beneath the bulge nucleotides (pink) to position the binuclear metal cluster (orange spheres) for splicing chemistry. The active site nonbridging oxygens are highlighted in black.

FIGURE 4.

Model of the active site of the group II intron during the second catalytic step (nucleophilic attack by the 3′-OH of the 5′ exon on the 3′ splice junction) of self-splicing showing biochemically implicated catalytic metal ions and the active site oxygens biochemically implicated in catalysis. The metal binding residues from the catalytic triad (violet) C358, G359, and bulge (pink) C377, and upper helical residue (pink) U375 from the O. iheyensi intron are shown. The cleavage site and the positioning of the 5′ exon by the EBS1 element were modeled and are not a part of the X-ray structure.