EFFECTS OF DIVALENT METAL-IONS ON INDIVIDUAL STEPS OF THE TETRAHYMENARIBOZYME REACTION

The Tetrahymena thermophila L-21 ScaI ribozyme utilizes Mg2+ to cataly ze a site-specific endonuclease reaction analogous to the first step o f self-splicing. To better understand the contribution of Mg2+ to ribo zyme activity, the Mg2+ concentration dependence of individual rate co nstants was examined at concentrations greater than those required for ribozyme folding (>2 mM; at 50 degrees C and pH 6.7). Analysis of met al ion inhibition of the chemical step of the reaction indicated that two Ca2+ ions compete with two Mg2+ ions involved in active site chemi stry. These Mg2+ ions are bound tightly to the E . S complex (K-d < 2 mM). The rate constant for association of the oligoribonucleotide subs trate (S) increased 12-fold from 2 to 100 mM Mg2+ and exhibited satura tion behavior, consistent with a single Mg2+ ion involved in S associa tion that binds to the free ribozyme with a K-d for Mg2+ of 15 mM. The preference for the divalent metal ion (Mg2+ congruent to Ca2+ > Ba2much greater than Sr2+) suggested that enhancing the rate constant of S association is not simply a function of ionic strength but is due to distinct metal ion binding Site. Even though Ca2+ does not support re action, the RNA substrate S was able to bind in the presence of Ca2+. Upon addition of Mg2+, S was cleaved without first dissociating. A mod el is proposed in which the inactive Ca2+ form of E . S is structurall y equivalent to the open complex along the reaction pathway, which has the RNA substrate bound but not (sic) into the active site. Weaker bi nding of S in Ca2+ was shown to result from an increase in the rate co nstant of S dissociation, leading to the proposal that a tight Mg2+ bi nding site or sites in the E . S complex contribute to the strong bind ing 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.