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

Swisher, Jennifer F.A.; Duarte, Carlos M.; Su, Linhui Julie; Pyle, Anna Marie

doi:10.1093/emboj/20.8.2051

Cited by 69 publications

(92 citation statements)

References 70 publications

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“…The NAIS experiments presented here clearly indicate some type of energetic communication between the D3IL and the D5 bulge, which is consistent with early DEPC footprinting studies that suggested molecular contacts between the D5 bulge and elements of D3 (Jestin et al 1997). The data also explain the striking solvent inaccessibility of the D3IL during hydroxyl radical footprinting experiments (Swisher et al 2001).…”

Section: D3 Supports a Complex Network Of Essential Interactionssupporting

confidence: 89%

“…It has been previously suggested that the D3IL forms a loop E-like motif in group IIA introns (Leontis and Westhof 1998b); however, it is not known whether this is true for other group II intron classes. Previous hydroxyl radical footprinting studies on IIB introns have shown that the D3IL is as highly internalized as D5 (Swisher et al 2001), suggesting that it is located within the catalytic core. In this study, we use a combination of NAIS and site-directed photo-cross-linking to identify molecular interaction partners between the D3IL and other regions of the intron.…”

Section: Introductionmentioning

confidence: 93%

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A conserved element that stabilizes the group II intron active site

Fedorova

Pyle²

2008

RNA

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The internal loop at the base of domain 3 (D3) is one of the most conserved and catalytically important elements of a group II intron. However, the location and molecular nature of its tertiary interaction partners has remained unknown. By employing a combination of site-directed photo-cross-linking and nucleotide analog interference suppression (NAIS), we show that the domain 3 internal loop (D3IL) interacts with the e-e9 duplex, which is an active-site element located near the 59-splice site in D1. Our data also suggest that the D3IL may interact with the bulge of D5, which is a critical active site component. The results of this and other recent studies indicate that the D3IL participates in a complex network of tertiary interactions involving e-e9, the bulge of D5 and J23, and that it helps to optimize active site architecture by supporting interactions among these catalytic motifs. Our results are consistent with the role of D3 as a catalytic effector that enhances intron reactivity through active site stabilization.

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Section: D3 Supports a Complex Network Of Essential Interactionssupporting

confidence: 89%

Section: Introductionmentioning

confidence: 93%

A conserved element that stabilizes the group II intron active site

Fedorova

Pyle²

2008

RNA

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“…Su), encoding exD135, was linearized with HindIII. This construct is a variant of pQL71 (Swisher et al 2001) containing a 293-nt-long 59 exon, D1, D3, and D5 as well as shortened hairpin-loop replacements of D2 and D4. All plasmids were transcribed as previously described (Pyle and Green 1994;Chin and Pyle 1995).…”

Section: Plasmids Templates and Rna Transcriptionmentioning

confidence: 99%

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

Lencastre¹,

Pyle²

2007

RNA

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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.

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“…On the other hand the solubility of RNA in dioxane-water mixtures is significantly reduced in comparison to the one in water, thus only mixtures containing up to 20% (v/v) of the organic solvent could be used in our NMR studies. Nevertheless, the permittivity of these solvent mixtures (ε = 61 -78) allows us to mimic to a certain extent the natural environment of this ribozyme domain, as D5 is located within the hydrophobic core of the three-dimensional architecture of the catalytically active RNA structure [62] being surrounded by the other five domains.…”

Section: <>mentioning

confidence: 99%