DiGIR2 is the group I splicing-ribozyme of the mobile twinribozyme intron Dir.S956-1, present in Didymium nuclear ribosomal DNA. DiGIR2 is responsible for intron excision, exon ligation, 3¢-splice site hydrolysis, and full-length intron RNA circle formation. We recently reported that DiGIR2 splicing (intron excision and exon ligation) competes with hydrolysis and subsequent full-length intron circularization. Here we present experimental evidence that hydrolysis at the 3¢-splice site in DiGIR2 is dependent on structural elements within the P9 subdomain not involved in splicing. Whereas the GCGA tetra-loop in P9b was found to be important in hydrolytic cleavage, probably due to tertiary RNA-RNA interactions, the P9.2 hairpin structure was found to be essential for hydrolysis. The most important positions in P9.2 include three adenosines in the terminal loop (L9.2) and a consensus kink-turn motif in the proximal stem. We suggest that the L9.2 adenosines and the kink-motif represent key regulatory elements in the splicing and hydrolytic reaction pathways.Keywords: Didymium iridis; group I intron; ribozyme hydrolysis; RNA processing; RNA structures.A highly conserved catalytic core is responsible for the self-splicing reactions of group I intron ribozymes [1]. The secondary structures of paired segments (P1-P10 and the optional P11-P17) are organized into three-dimensional domains were P4-P6 and P3-P9 form the catalytic core [2,3]. The available crystal structure of the Tetrahymena intron ribozyme core reveals an active site preorganized to catalysis [3], which appears to contain at least three metal ions directly involved in the reaction [4]. The group I introns can be divided into five main subgroups named IA, IB, IC, ID and IE [2,5]. The great majority of the more than 1200 group I introns recognized within nuclear rDNA belong to the group IC1 and group IE [6]. While the Tetrahymena intron (Tth.L1925) is a prototype group IC1 intron, the Didymium twin-ribozyme intron Dir.S956-1 (and its DiGIR2 derivative) is the best studied of the group IE introns.Group I intron splicing is initiated by binding of an exogenous guanosine (exoG) into the guanosine binding site (GBS). Here, exoG is positioned for attack at the 5¢-splice site (SS) and splicing proceeds through two consecutive transesterification steps. In addition to the essential exon splicing reactions, Tth.L1925 also catalyze hydrolytic cleavage at the 3¢-SS and the formation of truncated intron circles [1,7]. Hydrolytic cleavage at the 3¢-SS is initiated when the last intron nucleotide (TG) binds to the GBS prior to exoG. Splicing and hydrolysis are competing reactions leading to ligated exons and full-length intron circles, respectively [7].We have identified and examined an unusual category of self-splicing group I introns with a complex structural organization and function [8][9][10][11]. These twin-ribozyme introns consist of two distinct ribozymes (GIR1 and GIR2) and a homing endonuclease gene (HEG). The DiGIR2 ribozyme, encoded by the Didymium iridis twin-...