NMR structure of the 5′ splice site in the group IIB intron Sc.ai5γ—conformational requirements for exon–intron recognition

Abstract

A crucial step of the self-splicing reaction of group II intron ribozymes is the recognition of the 5 ′ exon by the intron. This recognition is achieved by two regions in domain 1 of the intron, the exon-binding sites EBS1 and EBS2 forming base pairs with the intron-binding sites IBS1 and IBS2 located at the end of the 5 ′ exon. The complementarity of the EBS1•IBS1 contact is most important for ensuring site-specific cleavage of the phosphodiester bond between the 5 ′ exon and the intron. Here, we present the NMR solution structures of the d3 ′ hairpin including EBS1 free in solution and bound to the IBS1 7-mer. In the unbound state, EBS1 is part of a flexible 11-nucleotide (nt) loop. Binding of IBS1 restructures and freezes the entire loop region. Mg 2+ ions are bound near the termini of the EBS1•IBS1 helix, stabilizing the interaction. Formation of the 7-bp EBS1•IBS1 helix within a loop of only 11 nt forces the loop backbone to form a sharp turn opposite of the splice site, thereby presenting the scissile phosphate in a position that is structurally unique.

The Role of Mg(II) in DNA Cleavage Site Recognition in Group II Intron Ribozymes

“…We could show by SPR measurements that Mg 2ϩ concentrations similar to the physiological concentration stabilize both the RNA⅐DNA and the RNA⅐RNA interaction strongly without altering the overall helical geometry. Control experiments did not reveal any changes in the fold or the flexibility of the unbound d3ЈEBS1 loop upon Mg 2ϩ addition (55). This is in line with the observation that Mg 2ϩ has little influence on the association of EBS1 and dIBS1.…”

“…2B). The chemical shifts of protons from the RNA stem are in excellent accordance with previously published ones for unbound d3ЈEBS1 (55). The sequential cross-peaks between U12 in the loop and G13 and G14 in EBS1 are very low in intensity (data not shown), probably due to an unusual geometry at residue G13.…”

“…Each imino proton in the d3Ј stem can be attributed to one resonance (black labels in Fig. 2A), their chemical shifts being very similar to the ones observed for the unbound d3ЈEBS1 (55), proving that the addition of dIBS1 does not interfere with the base pairing in the stem.…”

“…In the absence of their binding partner, dIBS1 is entirely unstructured, and d3ЈEBS1 forms a stable hairpin with an unstructured loop region (55). Upon dIBS1 binding to EBS1, the two form a short hybrid helix whose position relative to the stem is determined by the stacking interactions and putative hydrogen bonds between the single-stranded nucleotides surrounding EBS1.…”

“…We focus on a detailed analysis of the metal ion binding properties of the complex as determined by NMR spectroscopy and surface plasmon resonance (SPR). Because the same catalytic mechanism underlies intron-catalyzed DNA and RNA cleavage, we compare our data with the structure and metal ion binding of the analogous d3ЈEBS1⅐IBS1 homoduplex construct (55) and discuss common features relevant for stable binding of the target and for the recognition of the cleavage site.…”