Limits...
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
Model for DVI as the conformational switch for splicing. π-π’ mediates the transition between the two steps of catalysis. See text for details.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4197185&req=5

Figure 5: Model for DVI as the conformational switch for splicing. π-π’ mediates the transition between the two steps of catalysis. See text for details.

Mentions: During the first step, the bulged adenosine must be in close proximity to M1, M2, and the 5′ splice site in order to engage in nucleophilic attack. However, π-π’ places the bulged adenosine ~20 Å from the active site in the post-catalytic structure. Mutagenesis of π-π’ also has no significant effect on the first step of splicing (Fig. 3b), and is unlikely to be engaged for lariat formation. Furthermore, DII is likely to remain largely stationary during catalysis due to the strong anchoring effect of multiple tetraloop receptor interactions with this domain. Based on these observations, we propose that π-π’ is a dynamic interaction that toggles DVI between two different states to mediate the transition between the first and second steps of splicing (Fig. 5). In the first step, the bulged adenosine is engaged in the active site for nucleophilic attack at the 5′ splice site. At this stage, π-π’ exists in the “off” state where DII is disengaged from the base of DVI. The DVI helix would also presumably exist in a relaxed conformation due to the lack of constraint provided by π-π’. Following lariat formation, DVI likely engages in remodeling of its central internal loop adjacent to A615, causing helical compression to turn “on” the π-π’ interaction; thus sequestering the bulged adenosine away from the active site. A second possible model is that the base pairs between the G6 sequence (residues 588 to 593) and a pyrimidine-rich tract (612 to 614; 616 to 618) at the proximal side of the DVI stem rearrange to reposition the lariat phosphate and engage π-π’. Both models serve to empty the active site of the 5′ end and allow entry of the 3′ splice site, which is directly attached to the end of DVI. In fact, the primary function of the lariat may be to covalently attach to the 5′ end to provide an attachment point for this pulling action.


Crystal structure of a eukaryotic group II intron lariat.

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

Model for DVI as the conformational switch for splicing. π-π’ mediates the transition between the two steps of catalysis. See text for details.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Model for DVI as the conformational switch for splicing. π-π’ mediates the transition between the two steps of catalysis. See text for details.
Mentions: During the first step, the bulged adenosine must be in close proximity to M1, M2, and the 5′ splice site in order to engage in nucleophilic attack. However, π-π’ places the bulged adenosine ~20 Å from the active site in the post-catalytic structure. Mutagenesis of π-π’ also has no significant effect on the first step of splicing (Fig. 3b), and is unlikely to be engaged for lariat formation. Furthermore, DII is likely to remain largely stationary during catalysis due to the strong anchoring effect of multiple tetraloop receptor interactions with this domain. Based on these observations, we propose that π-π’ is a dynamic interaction that toggles DVI between two different states to mediate the transition between the first and second steps of splicing (Fig. 5). In the first step, the bulged adenosine is engaged in the active site for nucleophilic attack at the 5′ splice site. At this stage, π-π’ exists in the “off” state where DII is disengaged from the base of DVI. The DVI helix would also presumably exist in a relaxed conformation due to the lack of constraint provided by π-π’. Following lariat formation, DVI likely engages in remodeling of its central internal loop adjacent to A615, causing helical compression to turn “on” the π-π’ interaction; thus sequestering the bulged adenosine away from the active site. A second possible model is that the base pairs between the G6 sequence (residues 588 to 593) and a pyrimidine-rich tract (612 to 614; 616 to 618) at the proximal side of the DVI stem rearrange to reposition the lariat phosphate and engage π-π’. Both models serve to empty the active site of the 5′ end and allow entry of the 3′ splice site, which is directly attached to the end of DVI. In fact, the primary function of the lariat may be to covalently attach to the 5′ end to provide an attachment point for this pulling action.

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