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Crystal structure of a eukaryotic group II intron lariat.

Robart AR, Chan RT, Peters JK, Rajashankar KR, Toor N - Nature (2014)

Bottom Line: On the basis of structural and biochemical data, we propose that π-π' is a dynamic interaction that mediates the transition between the two steps of splicing, with η-η' serving an ancillary role.The structure also reveals a four-magnesium-ion cluster involved in both catalysis and positioning of the 5' end.Given the evolutionary relationship between group II and nuclear introns, it is likely that this active site configuration exists in the spliceosome as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
The formation of branched lariat RNA is an evolutionarily conserved feature of splicing reactions for both group II and spliceosomal introns. The lariat is important for the fidelity of 5' splice-site selection and consists of a 2'-5' phosphodiester bond between a bulged adenosine and the 5' end of the intron. To gain insight into this ubiquitous intramolecular linkage, we determined the crystal structure of a eukaryotic group IIB intron in the lariat form at 3.7 Å. This revealed that two tandem tetraloop-receptor interactions, η-η' and π-π', place domain VI in the core to position the lariat bond in the post-catalytic state. On the basis of structural and biochemical data, we propose that π-π' is a dynamic interaction that mediates the transition between the two steps of splicing, with η-η' serving an ancillary role. The structure also reveals a four-magnesium-ion cluster involved in both catalysis and positioning of the 5' end. Given the evolutionary relationship between group II and nuclear introns, it is likely that this active site configuration exists in the spliceosome as well.

Show MeSH
2Fo-Fc density for DVI in the pre-catalytic structure contoured at 1σ. The η-η’ interaction persists throughout the splicing reaction and is visible in the pre-catalytic state. The weaker density for the central region of DVI suggests a partially disordered, dynamic region with possible helical remodeling in the conserved internal loop during splicing. The general pattern of side-by-side packing of domains II and VI persists between the two steps. Catalytic triad mutation consisted of an AGC→GAU substitution.
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Figure 14: 2Fo-Fc density for DVI in the pre-catalytic structure contoured at 1σ. The η-η’ interaction persists throughout the splicing reaction and is visible in the pre-catalytic state. The weaker density for the central region of DVI suggests a partially disordered, dynamic region with possible helical remodeling in the conserved internal loop during splicing. The general pattern of side-by-side packing of domains II and VI persists between the two steps. Catalytic triad mutation consisted of an AGC→GAU substitution.

Mentions: DVI is proposed to engage in large-scale conformational changes between the two steps of splicing20,22. To test this model, a catalytic triad mutant (AGC→GAU) inactive for splicing was crystallized and solved at 7 Å (Extended Data Fig. 9). Strong electron density was observed for the η-η’ interaction between DII and DVI, indicating that this contact persists throughout both steps of splicing and that there is no large-scale change in DVI position. Therefore, a new model is required to explain how DVI mediates the transition between the two steps of splicing.


Crystal structure of a eukaryotic group II intron lariat.

Robart AR, Chan RT, Peters JK, Rajashankar KR, Toor N - Nature (2014)

2Fo-Fc density for DVI in the pre-catalytic structure contoured at 1σ. The η-η’ interaction persists throughout the splicing reaction and is visible in the pre-catalytic state. The weaker density for the central region of DVI suggests a partially disordered, dynamic region with possible helical remodeling in the conserved internal loop during splicing. The general pattern of side-by-side packing of domains II and VI persists between the two steps. Catalytic triad mutation consisted of an AGC→GAU substitution.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4197185&req=5

Figure 14: 2Fo-Fc density for DVI in the pre-catalytic structure contoured at 1σ. The η-η’ interaction persists throughout the splicing reaction and is visible in the pre-catalytic state. The weaker density for the central region of DVI suggests a partially disordered, dynamic region with possible helical remodeling in the conserved internal loop during splicing. The general pattern of side-by-side packing of domains II and VI persists between the two steps. Catalytic triad mutation consisted of an AGC→GAU substitution.
Mentions: DVI is proposed to engage in large-scale conformational changes between the two steps of splicing20,22. To test this model, a catalytic triad mutant (AGC→GAU) inactive for splicing was crystallized and solved at 7 Å (Extended Data Fig. 9). Strong electron density was observed for the η-η’ interaction between DII and DVI, indicating that this contact persists throughout both steps of splicing and that there is no large-scale change in DVI position. Therefore, a new model is required to explain how DVI mediates the transition between the two steps of splicing.

Bottom Line: On the basis of structural and biochemical data, we propose that π-π' is a dynamic interaction that mediates the transition between the two steps of splicing, with η-η' serving an ancillary role.The structure also reveals a four-magnesium-ion cluster involved in both catalysis and positioning of the 5' end.Given the evolutionary relationship between group II and nuclear introns, it is likely that this active site configuration exists in the spliceosome as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
The formation of branched lariat RNA is an evolutionarily conserved feature of splicing reactions for both group II and spliceosomal introns. The lariat is important for the fidelity of 5' splice-site selection and consists of a 2'-5' phosphodiester bond between a bulged adenosine and the 5' end of the intron. To gain insight into this ubiquitous intramolecular linkage, we determined the crystal structure of a eukaryotic group IIB intron in the lariat form at 3.7 Å. This revealed that two tandem tetraloop-receptor interactions, η-η' and π-π', place domain VI in the core to position the lariat bond in the post-catalytic state. On the basis of structural and biochemical data, we propose that π-π' is a dynamic interaction that mediates the transition between the two steps of splicing, with η-η' serving an ancillary role. The structure also reveals a four-magnesium-ion cluster involved in both catalysis and positioning of the 5' end. Given the evolutionary relationship between group II and nuclear introns, it is likely that this active site configuration exists in the spliceosome as well.

Show MeSH