Effects of Divalent Metal Ions on Individual Steps of the Tetrahymena Ribozyme Reaction
- 1 July 1997
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 36 (27), 8293-8303
- https://doi.org/10.1021/bi9700678
Abstract
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 degrees 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 (Kd < 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 Kd for Mg2+ of 15 mM. The preference for the divalent metal ion (Mg2+ congruent with 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.Keywords
This publication has 6 references indexed in Scilit:
- Replacement of the Conserved G.cntdot.U with a G-C Pair at the Cleavage Site of the Tetrahymena Ribozyme Decreases Binding, Reactivity, and FidelityBiochemistry, 1994
- Metal ion catalysis in the Tetrahymena ribozyme reactionNature, 1993
- How many catalytic RNAs? Ions and the Cheshire cat conjecture.The FASEB Journal, 1993
- Analysis of the role of phosphate oxygens in the group I intron from TetrahymenaJournal of Molecular Biology, 1992
- The molecular theory of polyelectrolyte solutions with applications to the electrostatic properties of polynucleotidesQuarterly Reviews of Biophysics, 1978
- tRNA Conformation and Magnesium BindingEuropean Journal of Biochemistry, 1975