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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.
Biochemistry. 1999 Aug 24; 38(34):10958-75.B

Abstract

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. 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, 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.

Authors+Show Affiliations

Department of Biochemistry, Stanford University, California 94305-5307, USA.No affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

10460151

Citation

Shan, S O., and D Herschlag. "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." Biochemistry, vol. 38, no. 34, 1999, pp. 10958-75.
Shan SO, Herschlag D. 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. Biochemistry. 1999;38(34):10958-75.
Shan, S. O., & Herschlag, D. (1999). 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. Biochemistry, 38(34), 10958-75.
Shan SO, Herschlag D. 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. Biochemistry. 1999 Aug 24;38(34):10958-75. PubMed PMID: 10460151.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Shan,S O, AU - Herschlag,D, PY - 1999/8/25/pubmed PY - 1999/8/25/medline PY - 1999/8/25/entrez SP - 10958 EP - 75 JF - Biochemistry JO - Biochemistry VL - 38 IS - 34 N2 - 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. 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, 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. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/10460151/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_ L2 - https://doi.org/10.1021/bi990388e DB - PRIME DP - Unbound Medicine ER -