Visualizing the solvent-inaccessible core of a group II intron ribozyme

Abstract

Group II introns are well recognized for their remarkable catalytic capabilities, but little is known about their three-dimensional structures. In order to obtain a global view of an active enzyme, hydroxyl radical cleavage was used to define the solvent accessibility along the backbone of a ribozyme derived from group II intron ai5gamma. These studies show that a highly homogeneous ribozyme population folds into a catalytically compact structure with an extensively internalized catalytic core. In parallel, a model of the intron core was built based on known tertiary contacts. Although constructed independently of the footprinting data, the model implicates the same elements for involvement in the catalytic core of the intron.

Fig. 1. Schematic secondary structure of the D135 ribozyme. D135 (heavy dark line) is comprised of domains 1, 3 and 5 of group II intron ai5γ; domains 2 and 4 have been replaced by hairpins and linker regions have been maintained. The 24 nt oligonucleotide substrate contains 17 nt of the 5′-exon sequence (including both intron binding sites, IBS1 and 2) and the first 7 nt of the intron.

Fig. 2. Active-site titration of the D135 ribozyme. Product formation was plotted as a function of time, in order to reveal the relative rates of the burst and steady state. The two phases of reaction were determined from the fit to equation 1 (Materials and methods). The fraction of active ribozyme molecules is determined from the intercept of the second phase with the y-axis. Reaction parameters reflect the average of three independent experiments.

Fig. 3. Equilibrium footprint of the D135 ribozyme. D135 samples were labeled on the 5′− (A) or 3′-end (B and C) to examine the solvent accessibility of all nucleotides in the ribozyme. In this manner, D1 (A and B), D3 (B) and D5 (C) were all visualized. Samples were incubated in the presence or absence of 100 mM MgCl₂, and then footprinted with potassium peroxynitrite (ONOOK; Materials and methods). T1 nuclease, which cleaves at G residues, provides a reference ladder (right-hand lanes).

Fig. 4. Effect of pH and KCl on footprinting patterns. Hydroxyl radical accessibility of D135 is compared at pH 6 and 7 (A) and in the presence and absence of 0.5 M KCl (B).

Fig. 5. Solvent accessibility map of D135. Regions of D135 that are protected from hydroxyl radical cleavage are mapped onto a secondary structural representation of the ribozyme. The degree of protection for each (Tables I and II) is indicated by the color code. Pairs of Greek letters correspond to known positions of tertiary interaction referred to in the text. Upper case Roman letters indicate domains of the ribozyme. Lower case Roman letters (and primes) indicate substructural helices within individual domains.

Fig. 6. Three-dimensional model of the minimal ai5γ core. The relative orientation of core D5 and D1 helices is shown. (A) D5 is shown in red, D1 helices that interact with the D5 binding face (including κ–κ′ and ζ–ζ′ interactions) are shown in green, while D1 helices that interact with the D5 chemistry face (including λ–λ′, ε–ε′ and EBS1–IBS1 interactions) are shown in yellow. In blue are helices that bridge these two faces, including α–α′. Greek letters indicate key long-range tertiary interactions. (B) A 90° rotation of the model around the x-axis, looking down from the top of the D5 tetraloop. This figure was generated using WebLab.