Deciphering the role of individual metal ions in RNA catalysis is a tremendous challenge, as numerous metal ions coat the charged backbone of a folded RNA. Metal ion specificity switch experiments combined with quantitative analysis may provide a powerful tool for probing specific metal ion-RNA interactions and for delineating the role of individual metal ions among the sea of metal ions bound to RNA. We show herein that Mn 2+ rescues the deleterious effect of replacing the 2′-OH of the guanosine nucleophile (G) by -NH 2 (G NH 2 ) in the reaction catalyzed by the Tetrahymena group I ribozyme (E), and the Mn 2+ concentration dependence suggests that a single metal ion is responsible for rescue. This provides strong evidence for a metal ion interaction with the 2′-moiety of G in this ribozyme (referred to as M C ), confirming and extending previous results in a bacteriophage group I intron [Sjögren, A.-S., Pettersson, E., Sjöberg, B.-M., and Strömberg, R. (1997) Nucleic Acids Res. 25, 648-654]. Toward understanding the >10 6 -fold catalytic contribution of the 2′-OH of G, we have determined the individual reaction steps affected by M C and quantitated these effects. Mn C 2+ has only a small effect on binding of G NH 2 to the free ribozyme or ribozyme‚oligonucleotide complexes that lack the reactive phosphoryl group. In contrast, Mn C 2+ increases the binding of G NH 2 to the ribozyme‚oligonucleotide substrate (E‚S) complex 20-fold and increases the binding of S to the E‚G NH 2 complex by the same amount. These and other observations suggest that M C plays an integral role in the coupled binding of the oligonucleotide substrate and the guanosine nucleophile. This metal ion may be used to align the nucleophile within the active site, thereby facilitating the reaction. Alternatively or in addition, M C may act in concert with an additional metal ion to coordinate and activate the 3′-OH of G. Finally, these experiments have also allowed us to probe the properties of this metal ion site and isolate the energetic effects of the interaction of this specific metal ion with the 2′-moiety of G.Divalent metal ions are essential for the structure and function of RNA. Numerous metal ions typically coat an RNA to neutralize the backbone charges and stabilize tertiary contacts, facilitating the folding of RNA into specific threedimensional structures (e.g., 2-19 and references cited therein). In addition to these structural roles, specific active site metal ions also directly participate in the chemical transformations catalyzed by many RNA enzymes (e.g., 20-26). However, the sea of metal ions bound to RNA presents an enormous challenge to deciphering the role of individual metal ions in RNA catalysis.Specific metal ion ligands have been identified by metal ion specificity switch experiments (20-32). These experiments exploit the preference of nitrogen and sulfur ligands to interact more strongly with soft metal ions, such as Mn 2+ , than with hard metal ions, such as Mg 2+ . The identification of metal ion ligands pro...