Modulation of individual steps in group I intron catalysis by a peripheral metal ion

Abstract

Enzymes are complex macromolecules that catalyze chemical reactions at their active sites. Important information about catalytic interactions is commonly gathered by perturbation or mutation of active site residues that directly contact substrates. However, active sites are engaged in intricate networks of interactions within the overall structure of the macromolecule, and there is a growing body of evidence about the importance of peripheral interactions in the precise structural organization of the active site. Here, we use functional studies, in conjunction with published structural information, to determine the effect of perturbation of a peripheral metal ion binding site on catalysis in a well-characterized catalytic RNA, the Tetrahymena thermophila group I ribozyme. We perturbed the metal ion binding site by site-specifically introducing a phosphorothioate substitution in the ribozyme's backbone, replacing the native ligands (the pro-R P oxygen atoms at positions 307 and 308) with sulfur atoms. Our data reveal that these perturbations affect several reaction steps, including the chemical step, despite the absence of direct contacts of this metal ion with the atoms involved in the chemical transformation. As structural probing with hydroxyl radicals did not reveal significant change in the three-dimensional structure upon phosphorothioate substitution, the effects are likely transmitted through local, rather subtle conformational rearrangements. Addition of Cd 2+ , a thiophilic metal ion, rescues some reaction steps but has deleterious effects on other steps. These results suggest that native interactions in the active site may have been aligned by the naturally occurring peripheral residues and interactions to optimize the overall catalytic cycle.

Functional Identification of Ligands for a Catalytic Metal Ion in Group I Introns

“…the red oxygen atom for Azoarcus residues U173 (U307) and the orange oxygen atom for Azoarcus residue G170 (A304)] and therefore seems unlikely to represent the missing M A ligand. This analysis supports the model that the effects observed upon introduction of a sulfur atom at the R P position of A304 are due to a rearrangement of the active site upon thio substitution, as described in other systems (22,(49)(50)(51) Phosphorothioate substitution at position U307R P , a position implicated in binding a peripheral metal ion referred to as M E , shifts the rescue profile to higher Cd 2+ concentrations. Possible reasons for this effect have been discussed in previous work (22).…”

“…With the exception of the A304R P and the U307R P ribozymes, Cd 2+ affected the reactivity of the ribozymes less than 3-fold. The stimulatory effect on the U307R P ribozyme was previously investigated and ascribed to a peripheral metal ion, M E , that interacts with the introduced sulfur atom on the ribozyme and modulates several reaction steps (22). There was also a small stimulatory effect on the A304R P ribozyme (2.5-fold), even though the substituted oxygen atom was not implicated in functional contacts with metal ions from structural data.…”

Exploring purine N7 interactions via atomic mutagenesis: The group I ribozyme as a case study

“…(Adams et al 2004;Golden et al 2005;Strobel 2005, 2007) but not in those derived from crystal of the Tetrahymena group I ribozyme (Guo et al 2005), probably because of the particular truncation of the crystallized construct. The contacts between this Mg 2+ ion and the pro-R P phosphoryl oxygens of residues 307 and 308 are supported by functional studies on the Tetrahymena group I ribozyme (Forconi et al 2007). …”

“…The Hoogsten face of guanosine is located near a cavity in the group I active site that can accommodate several water molecules (Adams et al 2004;Golden et al 2005;Stahley and Strobel 2005;Forconi et al 2007), and recent studies have suggested that deaza substitutions in nucleobases are associated with significant water and cation loss (Ganguly et al 2007(Ganguly et al , 2009(Ganguly et al , 2010.…”

Insights into functional modulation of catalytic RNA activity

“…that facilitate this folding (87). It was recently shown that for the organization of the functional architecture of the Tetrahymena group I intron active site, a number of interactions, mediated by a peripheral metal ion, are important (89). This metal ion can affect differentially several steps of the catalytic process, indicating that its role is not precisely described as structural or catalytic.…”