Self-splicing group II introns belong to the class of large ribozymes. Recognized as an independent class only in 1982 (1
), they are found primarily in organellar genes of plants, fungi and lower eukaryotes, but also in many bacteria (2
). It is thought that they were crucial in modeling the genome of almost all terrestrial life forms: a common ancestry has been proposed for group II introns and most nuclear introns, components of the telomerase and the abundant LINE–element form of transposons (4
), as well as the eukaryotic spliceosome (5–9
). Moreover, they have been found to be able to reinsert into RNA and DNA via a retro-homing mechanism (10–12
), thus being mobile genetic elements responsible for the continuous diversification of different genomes.
Group II introns as all other RNA molecules are characterized by a secondary structure usually stabilized by monovalent metal ions, whereas the tertiary structure requires the presence of divalent metal ions (13
). The latter are eventually also involved in catalysis (14–17
). Despite a lack of primary structure conservation, all group II introns present a similar secondary structure, consisting of six domains originating from a central wheel (2
). Domain 1 represents the largest domain. It is an independent folding unit (20
) and provides a scaffold for the other domains to dock. Domain 2 and 3 stabilize the folding and enhance the catalytic activity of the whole intron, respectively. Domain 4 can contain an open reading frame encoding for a maturase protein useful for intron activity in vivo
). Domain 5 is the genetically most conserved one and shares many features with the spliceosomal RNA U6 (22
). It represents, together with domain 1, the minimal core to enable catalysis (3
). Finally, domain 6 contains the bulged adenosine implicated as nucleophile in the first step of splicing (23
). Several studies have been devoted to understanding and describing both the folding and the catalytic mechanism of group II introns. One of the best studied systems is the Saccharomyces cerevisiae
.ai5γ from baker’s yeast (24–29
In particular, a shorter version of the whole intron containing domains 1, 3 and 5 (D135) has been extensively used for folding studies (28–32
). The folding of large RNAs usually involves a rapid collapse on metal ion addition (33
). The collapse can be unspecific or can be initiated by the formation of specific RNA substructures. Pyle et al.
) showed that D135 folds in an apparent two state model: the first stage is slow (in the order of seconds) and requires the formation of specific structural features within D1. This stage is then followed by the fast docking of all the other domains. Both stages require above-physiological Mg(II) concentrations in vitro
, the first one has a KD,Mg
of ~13 mM (21
), the second of 20–40 mM (30
A small subregion in the central core of D1 comprising the three-way junction around the κ motif (25
) and an 11-nt tetraloop receptor (ζ) was proposed to act as folding control element (D1κζ, ) (24
). In the presence of Mg(II), this region adopts a specific structure, which allows the whole intron to attain the active native state (29
). Moreover, it was recently shown that Mss116p, a DEAD box protein, which promotes folding in vivo
substituting in part for the Mg(II) (35
) acts by stabilizing this small substructure (37
). In a later stage of folding, D1κζ also offers the docking site for D5, as supported by biochemical studies and a recent crystal structure of a group IIC intron from Oceanobacillus iheyensis
Figure 1. (a) Predicted secondary structure of the group II intron Sc.ai5γ from S. cerevisiae (the κ-ζ region studied in this work is highlighted). (b) The wild-type κ-ζ region (top) and the 49 nucleotide construct used for (more ...)
We have now investigated the structure of D1κζ by means of nuclear magnetic resonance (NMR) spectroscopy, and demonstrate the central role of Mg(II) in its stabilization. Our data show that the helices next to the three-way junction are destabilized in the absence of Mg(II). Coordination of Mg(II) leads to a rigidification of the central core of this substructure, resulting in the coaxial stacking of two helices as was suggested by Costa et al.
) from comparative sequence analysis.
The bulged nucleotides of the D1κζ junction form a classical GAAA tetraloop motif interrupted between the third and the forth nucleotide by a short helix. In addition, adenosines in this motif are engaged in A-minor interactions with the adjacent helix. Interestingly, this A-minor motif is not present in the only available crystal structure of a group IIC intron (38–40
), where the two adenines are interacting with the docked D5.
Coaxial stacking of helices and A-minor motifs have been recognized as among the most important interactions governing RNA tertiary structure (42
) and are here combined together within this small subregion. Helices next to multibranch junctions tend to maximize coaxial stacking to reduce overall folding free energy (43
), and the stacking has been shown to depend on di- or multivalent ions (44
). Our detailed NMR data allow us to locate a possible binding pocket at the junction that explains the requirement of multivalent metal ions for stabilization of this region. Which helices in a junction stack and how they are oriented is strongly influenced by the bulged nucleotides. Geary et al.
) have highlighted the versatility of A-minor interactions in determining the architecture of junctions and the relative orientation of the helices. To the best of our knowledge, the structural arrangement of the A-minor interactions in the three-way junction present in D1κζ has not been described previously.
This NMR solution structure of D1κζ adds to the scarce structural data on group II introns (23
). Moreover, we reveal κ to be a flexible, bimodal structure, which keeps its characteristic fold both in the context of a minimal A-minor junction motif to the adjacent helix (as observed in the D1κζ construct), as well as in the tertiary interaction with D5 (in the full group II intron).