Effects of Divalent Metal Ions on Individual Steps of the Tetrahymena Ribozyme Reaction

• Published in: Biochemistry (Easton) 1997-07, Vol.36 (27), p.8293-8303
• Main Authors: McConnell, Timothy S, Herschlag, Daniel, Cech, Thomas R
• Format: Article
• Language: English
• Subjects: Animals; Barium - pharmacology; Binding Sites; Binding, Competitive; Calcium - metabolism; Calcium - pharmacology; Catalysis; Cations, Divalent; Kinetics; Magnesium - metabolism; Magnesium - pharmacology; Nucleic Acid Conformation; Oligoribonucleotides - metabolism; Osmolar Concentration; RNA Splicing; RNA, Catalytic - metabolism; Strontium - pharmacology; Tetrahymena thermophila - enzymology; Tetrahymena thermophila - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9700678

The Tetrahymena thermophila L-21 ScaI ribozyme utilizes Mg2+ to catalyze a site-specific endonuclease reaction analogous to the first step of self-splicing. To better understand the contribution of Mg2+ to ribozyme activity, the Mg2+ concentration dependence of individual rate constants was examined at concentrations greater than those required for ribozyme folding (>2 mM; at 50 °C and pH 6.7). Analysis of metal ion inhibition of the chemical step of the reaction indicated that two Ca2+ ions compete with two Mg2+ ions involved in active site chemistry. These Mg2+ ions are bound tightly to the E·S complex (K d < 2 mM). The rate constant for association of the oligoribonucleotide substrate (S) increased 12-fold from 2 to 100 mM Mg2+ and exhibited saturation behavior, consistent with a single Mg2+ ion involved in S association that binds to the free ribozyme with a K d for Mg2+ of 15 mM. The preference for the divalent metal ion (Mg2+ ≅ Ca2+ > Ba2+ ≫ Sr2+) suggested that enhancing the rate constant of S association is not simply a function of ionic strength, but is due to a distinct metal ion binding site. Even though Ca2+ does not support reaction, the RNA substrate S was able to bind in the presence of Ca2+. Upon addition of Mg2+, S was cleaved without first dissociating. A model is proposed in which the inactive Ca2+ form of E·S is structurally equivalent to the open complex along the reaction pathway, which has the RNA substrate bound but not docked into the active site. Weaker binding of S in Ca2+ was shown to result from an increase in the rate constant of S dissociation, leading to the proposal that a tight Mg2+ binding site or sites in the E·S complex contribute to the strong binding of S. In summary, the data provide evidence for four functions for bound Mg2+ ions in the catalytic cycle:  one increases the rate of RNA substrate binding, one or more decrease the rate of dissociation of S, and two are involved in the chemical step.