Crystal structure of a group II intron in the pre-catalytic state

Abstract

Group II introns are self-splicing catalytic RNAs that are thought to be ancestral to the spliceosome. Here we report the 3.65-Å crystal structure of the group II intron from Oceanobacillus iheyensis in the pre-catalytic state. The structure reveals the conformation of the 5' splice site in the catalytic core and represents the first structure of an intron prior to the first step of splicing.

Figure 1

The pre-catalytic structure of the group II intron. a) The structure of the pre-catalytic state reveals the position of the 5′ splice site (junction between green and blue) in the center of the molecule. b) Close-up view of the 5′ splice site reveals a sharp kink in the backbone, which is positioned near the bulge and catalytic triad of domain V (red). c) F_o-F_c density for the 5′ splice site contoured at 3σ. The F_o-F_c density map was calculated using a model deleted for intron nucleotides 1–5 and the 5′ exon in order to avoid model bias.

Figure 2

Theoretical model for the complete group II intron splicing pathway. a) The 5′ splice site is kinked immediately before the onset of catalysis. The kink is positioned in close proximity to the catalytic metal ions (M1 and M2). A ribose 2′-OH group or water molecule (not depicted) is activated for nucleophilic attack and cleaves the splice site. Residue 288 is not shown because there is no electron density for the base of this nucleotide in the pre-catalytic structure. b) The 3′ splice site is positioned in the active site through its interaction with EBS3 and the γ nucleotide. These tertiary interactions cause the 3′ splice site to also adopt a sharp kink which presents the scissile phosphate to the active site metal ions. The 3′-OH (shown in stick format) of the 5′ exon is in a position to coordinate to M1 as well as to the 3′ splice site (coordination indicated by black lines). G1 and U2 are not shown due to the fact that these nucleotides must depart before the 3′ splice site enters the active site. c) The 3′ splice site is cleaved, the exons ligated, and the product adopts a “relaxed” conformation prior to release by the intron. Abbreviation: J2/3 - junction sequence between domains II and III.