Catalytic Properties of the Intron RNA

Group II intron RNAs are ribozymes capable of self-splicing in vitro via two sequential transesterification reactions that result in the formation of an intron lariat (Fig. lb). Chemically, the splicing reactions are the same as those of spliceosomal introns, which are believed to be evolutionary descendants

Fig. 1. Group II intron RNA secondary structure and splicing mechanism, a Conserved RNA secondary structure. The structure consists of six double-helical domains (DI-DVI) radiating from a central wheel; subdomains are indicated by lowercase letters (e.g., DIVa). The ORF is encoded within DIV (dotted loop), and DIVa is a high-affinity binding site for the IEP. The locations of the Shine-Dalgarno (SD) sequence, AUG initiation codon, and UAA termination codon are indicated. Greek letters and gray shading indicate sequences involved in tertiary interactions (light gray lines). EBS and IBS denote exon- and intron-binding sites, respectively. The structure shown is for the L. lactis Ll.LtrB intron (Mills et al. 1996; Shearman et al. 1996). b Splicing mechanism. In the first transesterification, nu-cleophilic attack at the 5'-splice site by the 2' OH of a bulged A-residue in DVI results in cleavage of the 5'-splice site coupled to formation of lariat intermediate. In the second transesterification, nucleophilic attack at the 3'-splice site by the 3' OH of the cleaved 5' exon results in exon ligation and release of the intron lariat, c EBS/IBS and S-S' interactions between the Ll.LtrB intron and flanking 5'- and 3'-exon sequences in unspliced precursor RNA. The 5' and 3' exons are indicated by black and gray shading, respectively. SS, splice site of group II introns (Michel and Ferat 1995). To catalyze splicing, group II intron RNAs, like protein enzymes, fold into a specific three-dimensional structure that forms an active site (Qin and Pyle 1998). For group II introns, the active site is thought to include Mg2+ ions bound at specific positions in DV, in combination with parts of DI (Sigel et al. 2000; Gordon and Piccirili 2001). The active site aligns and activates specific chemical bonds, including the 2' OH of the branch-point A in DVI and the phosphodiester bonds at the 5'- and 3'-splice sites, which must be broken and joined during splicing. The folding

Fig. 2. Group II intron-encoded proteins. Protein coding regions are shown as rectangles, with different shading, indicating conserved regions or domains. Exons (E) are black boxes. Protein domains are: RT, with conserved sequence blocks RT-0 to -7 indicated below and delineated above; X, associated with maturase activity; D, DNA-binding; and En, DNA endonuclease. a S. cerevisiae mtDNA introns coxl-II and -12. b L. lactis Ll.LtrB intron. c S. meliloti Rmlntl intron, which encodes a protein lacking the En domain. D? in Rmlntl indicates that the function of this region has not been established

Fig. 2. Group II intron-encoded proteins. Protein coding regions are shown as rectangles, with different shading, indicating conserved regions or domains. Exons (E) are black boxes. Protein domains are: RT, with conserved sequence blocks RT-0 to -7 indicated below and delineated above; X, associated with maturase activity; D, DNA-binding; and En, DNA endonuclease. a S. cerevisiae mtDNA introns coxl-II and -12. b L. lactis Ll.LtrB intron. c S. meliloti Rmlntl intron, which encodes a protein lacking the En domain. D? in Rmlntl indicates that the function of this region has not been established of the intron RNA to form the active site involves both intra- and interdomain tertiary interactions, of which over a dozen have been identified (Fig. la; IBS1-3/EBS1-3, a-a' to X-X'). NMR and X-ray crystal structures have been determined for DV and DV+DVI, respectively (Zhang and Doudna 2002; Sigel et al. 2004), and structural models have been proposed for the active site (Costa et al. 2000; Swisher et al. 2001).

An important feature of the group II intron splicing reaction is that 5'- and 3'-exon sequences flanking the splice sites are bound at the active site by base-pairing interactions with specific sequence elements in DI (Fig. la,c; Michel and Ferat 1995; Qin and Pyle 1998; Costa et al. 2000). The sequence elements EBS1 and EBS2 (exon-binding sites 1 and 2), located in two different stem-loops in DI, base pair with 5'-exon sequences IBS1 and IBS2 (intron-bind-ing sites 1 and 2), directly upstream of the 5'-splice site. The 3' exon is recognized by another short pairing, which differs between group IIA and IIB introns. In group IIA introns, the sequence 6, adjacent to EBSl,base pairs with the first few nucleotide residues of the 3' exon (6'; Fig. lc), while in group IIB

and probably IIC introns, a different sequence element, EBS3, base pairs with the 3' exon (IBS3; not shown). Because the transesterification reactions catalyzed by the intron RNA are reversible, the same base-pairing interactions between the intron and the 5' and 3' exons are required not only for RNA splicing, but also for reverse splicing into RNA, or into DNA target sites during intron mobility. This feature enables the facile engineering of homing endonucleases with different target specificities (see below).

Finally, although group II introns are intrinsically catalytic, most if not all require proteins to help fold the RNA into the active structure for efficient splicing (reviewed in Lambowitz et al. 1999; Lehmann and Schmidt 2003). In the case of ORF-containing group II introns, the key splicing factor is the IEP, which binds specifically to its own intron RNA and stabilizes the catalytical-ly active structure ("maturase" activity; Carignani et al. 1983; Saldanha et al. 1999; Matsuura et al. 2001; Noah and Lambowitz 2003). The dependence of mobile group II introns on a single major splicing factor encoded within the intron enables them to function in different organisms. The L. lactis Ll.LtrB intron, for example, is spliced efficiently in both its natural host and a number of other Gram-positive or Gram-negative bacteria (Mills et al. 1996; Shearman et al. 1996; Matsuura et al. 1997; Belhocine et al. 2004; Staddon et al. 2004).

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