Three essential and conserved regions of the group II intron are proximal to the 5′-splice site

Abstract

Despite the central role of group II introns in eukaryotic gene expression and their importance as biophysical and evolutionary model systems, group II intron tertiary structure is not well understood. In order to characterize the architectural organization of intron ai5g, we incorporated the photoreactive nucleotides s 4 U and s 6 dG at specific locations within the intron core and monitored the formation of cross-links in folded complexes. The resulting data reveal the locations for many of the most conserved, catalytically important regions of the intron (i.e., the J2/3 linker region, the IC1(i-ii) bulge in domain 1, the bulge of D5, and the 59-splice site), showing that all of these elements are closely colocalized. In addition, we show by nucleotide analog interference mapping (NAIM) that a specific functional group in J2/3 plays a role in first-step catalysis, which is consistent with its apparent proximity to other first-step components. These results extend our understanding of active-site architecture during the first step of group II intron self-splicing and they provide a structural basis for spliceosomal comparison.

A conserved element that stabilizes the group II intron active site

“…The same studies show that D5 bulge nucleotide A838 interacts with C4 of the e-e9 motif (de Lencastre et al 2005;de Lencastre and Pyle 2008). This is remarkable in light of the present study, in which the same motif is implicated through cross-linking and NAIS methods to interact with the D3IL.…”

“…A contact between the D3IL and e-e9 is particularly significant in light of recent studies demonstrating that e-e9 is arranged in close proximity to the bulge of D5 and the J23 linker (de Lencastre et al 2005;de Lencastre and Pyle 2008), which are two of the most important activesite components in a group II intron. By extension, an interaction between the internal loop of D3 and e-e9 would be expected to play an important role in stabilizing the active-site structure, which is consistent with the fact that the D3IL stimulates the chemical rate constant for catalysis (Bachl and Schmelzer 1990;Boudvillain and Pyle 1998).…”

DEAD-box protein facilitated RNA folding in vivo

“…In addition, residues involved in the ε-ε' and λ-λ' contacts are spatially close to the D5 dinucleotide bulge and J2/3, both of which are known key players in active-site organization. 47,56 In the O. iheyensis group II intron crystal structure 41 the invariant G5 (ε) interacts with A376 (A838 in ai5γ), which is extruded from the D5 helix. C377 (C839 in ai5γ), in contrast, stacks on top of G288 (G588 in ai5γ) and C289 (A589 in ai5γ) in J2/3, forming a triple-helix with the catalytic triad (C358-C360 in the Oi.…”

The structure and function of catalytic RNAs

“…D1 contains nucleotides that are essential for the recognition of both exons and the branch-site nucleophile [53] . D2 and D3 are essential components for the full catalytic activity, and the junction regions between them (J2/3) is part of the active center [54] . D4 usually encodes a large open reading frame which functions in intron mobility, and functions little in ribozyme folding or catalysis [55] .…”