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

Abstract

Many enzymes use metal ions within their active sites to achieve enormous rate acceleration. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). The three-dimensional arrangement determined by X-ray crystallography provides a powerful starting point for identifying ground state interactions, but only functional studies can establish and interrogate transition state interactions. The Tetrahymena group I ribozyme is a paradigm for the study of RNA catalysis, and previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified catalytic metal ions making five contacts with the substrate atoms. Here, we have combined atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to establish transition state ligands on the ribozyme for one of the catalytic metal ions, referred to as M A . We identified the pro-S P oxygen atoms at nucleotides C208, A304, and A306 as ground state ligands for M a , verifying interactions suggested by the Azoarcus crystal structures. We further established that these interactions are present in the chemical transition state, a conclusion that requires functional studies, such as those carried out herein. Elucidating these active site connections is a crucial step toward an in-depth understanding of how specific structural features of the group I intron lead to catalysis.Phosphoryl transfer is a ubiquitous reaction in biology, yet it is extremely slow in solution (1,2). To accelerate this class of reactions to an extent compatible with life, many enzymes have evolved active sites that contain metal ions. To understand how enzymes utilize the catalytic power of metal ions, we must identify individual metal ions, define their coordination † This work was supported by a grant from the NIH (GM 49243) to D.H. and by a grant from the Howard Hughes Medical Institute to J.A.P.© 2008 American Chemical Society *Address correspondence to this author at the Department of Biochemistry, Beckman Center, B400, Stanford University. . E-mail: herschla@stanford.edu . SUPPORTING INFORMATION AVAILABLESupporting text with references to metal ion rescue experiments in protein and RNA enzymes and discussion of possible complications in TFA analysis; Table S1, providing the values of the parameters in Scheme 3 for the M B rescue of CUCG 3′S A; Table S2, providing the values of the parameters in Scheme 3 for the M C rescue of G N ; Figures S1, S2, S3, and S5, providing the data prior to normalization associated with Figure 4B,C, Figure 5B, and Figure 6B, respectively; and Figure S4, providing the comparison of M B rescue of CUCG 3′S A using two different substrates, −1r,dP and −1d,rP, for the wt and U307S P ribozymes. This material is available free of charge via the Internet at http://pubs.acs.org NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 October 6. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-P...

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

“…1D; Stahley and Strobel 2005;Lipchock and Strobel 2008). Models for this discrepancy have been described (Discussion; see Hougland et al 2006) and we note that the remaining interactions inferred from the functional and structural data are in excellent agreement (Hougland et al 2006;Forconi et al 2008Forconi et al , 2009 …”

“…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).…”

“…We did not use this structure to model (E•S•G) O because one metal ion, presumably M C , is observed in the active site. We speculate that metal ion binding to site A may have been disrupted from mutating A304, a M A ligand (Forconi et al 2008), to G or from removing helices P1, P2, P9.1, and P9.2 from the intron.…”

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.…”

Metal-ion rescue revisited: Biochemical detection of site-bound metal ions important for RNA folding

“…Metal-ion rescue experiments, first used to delineate metal-ion substrate interactions in protein enzyme active sites (Jaffe and Cohn 1978;Darby and Trayer 1983;Lee and O'Sullivan 1985;Pecoraro et al 1985), have been expanded in ribozyme studies to provide a powerful strategy to identify individual metal ions, define their ligands, and assign their roles in catalysis (Piccirilli et al 1993;Chen et al 1997;Sontheimer et al 1997;Weinstein et al 1997;Shan et al 1999;Yoshida et al 1999;Hougland et al 2005;Gordon et al 2007;Forconi et al 2008;Forconi and Herschlag 2009 , and Zn 2+ to coordinate oxygen vs. sulfur or nitrogen ligands. As Mg 2+ coordinates these ligands more weakly than it coordinates oxygen, sulfur or nitrogen substitution of an oxygen ligand can disrupt the Mg 2+ interaction, leading to deleterious effects on activity.…”