Ribozyme Catalysis from the Major Groove of Group II Intron Domain 5

Abstract

The most highly conserved nucleotides in D5, an essential active site component of group II introns, consist of an AGC triad, of which the G is invariant. To understand how this G participates in catalysis, the mechanistic contribution of its functional groups was examined. We observed that the exocyclic amine of G participates in ground state interactions that stabilize D5 binding from the minor groove. In contrast, each major groove heteroatom of the critical G (specifically N7 or O6) is essential for chemistry. Thus, major groove atoms in an RNA helix can participate in catalysis, despite their presumed inaccessibility. N7 or O6 of the critical G could engage in critical tertiary interactions with the rest of the intron or they could, together with phosphate oxygens, serve as a binding site for catalytic metal ions.

Influence of decreased solvent permittivity on the structure and magnesium(II)-binding properties of the catalytic domain 5 of a group II intron ribozyme

“…This nucleotide is highly conserved, forming a wobble pair with U32, and part of the so-called AGC or catalytic triad. Its minor groove site is involved in tertiary contacts whereas the major groove is implicated to harbour the catalytic site [68]. Our NOESY data under these conditions does not support a higher dynamics or even flipping out of G3, as it has been suggested previously [69], but shows an intact GU wobble pair.…”

The intramolecular stem-loop structure of U6 snRNA can functionally replace the U6atac snRNA stem-loop

“…In spite of the high conservation of the structure of this element and the apparent conservation of its function as shown by these studies, we do not have a clear idea of what its function might be in the spliceosome+ Its location near the sites of splicing chemistry have led to speculation that it plays a role in the active site of the spliceosome (reviewed in Nilsen, 1998;Collins & Guthrie, 2000)+ Several groups have suggested parallels with features of various ribozymes including the hairpin ribozyme (Tani & Ohshima, 1991;Sun & Manley, 1995) and domain 5 of group II self-splicing introns (see discussions in Sun & Manley, 1997;Costa et al+, 1998;Nilsen, 1998)+ The group II domain 5 comparison is particularly interesting+ A recent revision of the proposed structure of the domain 5 stem-loop has emphasized the similarity between it and the U6 (or U6atac) intramolecular stem-loop (Costa et al+, 1998)+ A phosphorothioate modification-interference study of domain 5 in self-splicing identified a phosphate group in the bulge region as important for splicing catalysis (Chanfreau & Jacquier, 1994)+ This phosphate is located in a very similar position to one identified as important for U6 snRNA function in in vitro pre-mRNA splicing in both yeast (Fabrizio & Abelson, 1992) and nematodes (Yu et al+, 1995)+ Recent investigations of both phosphorothioate diastereomers at this position in yeast U6 snRNA have revealed a metal ion specificity switch for the first step of splicing (Yean et al+, 2000)+ This suggests that U6 snRNA participates in the catalysis of splicing through metal ion coordination and places this stem-loop element at or very near the catalytic center of the spliceosome+ Pyle's group has also suggested that the bulge and lower stem regions of domain 5 serve to position a critical metal ion for group II splicing (Konforti et al+, 1998)+ Such a function would mainly involve the positioning of phosphate groups to coordinate the metal and would be compatible with many but perhaps not all base paired sequences within the stem region+ Basespecific interactions would be limited to residues in unpaired regions and functional groups in the major and minor groves of the helical regions+ Such a function would fit with the apparent tolerance of the stem regions of U6atac snRNA for many but not all substitutions that maintain base pairing+…”

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

“…Also, DV with the bulged nucleotides (purple in Fig. 7) and the catalytic triad of DV, which are known to play a major role in catalysis (Chanfreau and Jacquier 1994;Konforti et al 1998;Sigel et al 2004), is shown. The intron was crystallized in the presence of Ca 2+ ions, which inhibit the splicing reaction (Erat and Sigel 2008) and represent the conformation before the first splicing step.…”