Functional Identification of Catalytic Metal Ion Binding Sites within RNA

Abstract

The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (MA, MB, and MC) that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-S P phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for MC. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.

Metal binding and substrate positioning by evolutionarily invariant U6 sequences in catalytically active protein-free snRNAs

“…In well-studied ribozymes, site-bound metal ions play critical roles in catalysis and it has been possible to deduce their location using biochemical methods (for example, see Shan et al 1999;Sigel et al 2000;Hougland et al 2005;Forconi et al 2008;Frederiksen and Piccirilli 2009). As our data are consistent with the involvement of divalent cations in catalysis of the snRNA-mediated splicing, we asked whether we could determine the existence and location of sitespecifically bound divalent cations in the U6/U2 complex used in our assays.…”

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

“…5A,B). Functional data (Forconi et al 2010) suggest that this hydrogen bond is made in (E•S•G) C but not (E•S•G) O and we speculate that the absence of this contact releases the adjacent C262 residue, a ligand for M C (Hougland et al 2005). Such a change might allow M C to sample alternative positions (Fig.…”

“…By comparing the affinities of metal ions providing rescue of substitutions at different positions, one can determine whether these interactions are mediated by the same metal ion or by distinct metal ions (Shan et al 1999a;Wang et al 1999;Christian 2005;Hougland et al 2006;Frederiksen and Piccirilli 2009). Such analyses and follow-up experiments can lead to detailed maps of functional interactions (Shan et al 1999a;Wang et al 1999;Christian et al 2000;Gordon et al 2000;Gordon and Piccirilli 2001;Hougland et al 2005;Forconi et al 2008;Ward and Derose 2012;Fica et al 2013).…”

Influence of decreased solvent permittivity on the structure and magnesium(II)-binding properties of the catalytic domain 5 of a group II intron ribozyme

“…In the case of ribozymes divalent metal ions are in addition implicated in catalysis [7,[13][14][15] and in most ribozyme structures they are found close to the catalytic core. It is relevant to mention in this context that labile and biologically relevant metal ions can coordinate to RNA in close neighbourhood to each other [16,17] and even to the same nucleobase [18].…”