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Published August 1, 1999 | public
Journal Article

Probing the Role of Metal Ions in RNA Catalysis:  Kinetic and Thermodynamic Characterization of a Metal Ion Interaction with the 2'-Moiety of the Guanosine Nucleophile in the Tetrahymena Group I Ribozyme


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_(NH2)) 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 MC), 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 MC and quantitated these effects. Mn_C^(2+) has only a small effect on binding of G_(NH2) to the free ribozyme or ribozyme·oligonucleotide complexes that lack the reactive phosphoryl group. In contrast, Mn_C^(2+) increases the binding of G_(NH2) to the ribozyme·oligonucleotide substrate (E·S) complex 20-fold and increases the binding of S to the E·G_(NH2) complex by the same amount. These and other observations suggest that MC 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, MC 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.

Additional Information

© 1999 American Chemical Society. Received February 17, 1999; Revised Manuscript Received May 14, 1999. This work was supported by NIH Grant GM49243 to D.H. We are grateful to members of the Piccirilli group for helpful discussions and technical support, F. Eckstein for the gift of 2'-aminoguanosine, L. Beigelman for oligonucleotide substrates, G. Narlikar for initial results and intellectual input, and members of the Herschlag group for helpful comments on the manuscript.

Additional details

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October 18, 2023