Effects of Mg2+ and the 2‘ OH of Guanosine on Steps Required for Substrate Binding and Reactivity with the Tetrahymena Ribozyme Reveal Several Local Folding Transitions

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Abstract
Transient kinetic studies with fluorescence detection were used to determine the effects of Mg2+ concentration and the 2' OH group of guanosine monophosphate, prG, substrate on various steps in the transesterification reaction of prG with 5' pyrene-labeled oligonucleotides as catalyzed by the L-21 ScaI ribozyme. The effect of increasing Mg2+ from 5 to 10 mM on the rate constants of association and dissociation of 5' pyrene-labeled CUCUA at 15 degrees C was measured. The rate constant of association increases about 3-fold to (8.7 +/- 0.7) x 10(6) M-1 s-1 at 10 mM Mg2+. The rate constant for dissociation is 25 +/- 4 s-1 at 10 mM Mg2+, within experimental error of the rate constant of 17 +/- 5 s-1 measured at 5 mM Mg2+. This Mg2+ dependence is attributed to nonspecific binding of Mg2+ to the duplex helix. In the absence of prG, no docking of substrate is observed. The effect of Mg2+ concentration on rates for docking of 5' pyrene-labeled substrate, pyrCCUCUA, were measured at [Mg2+] >/= 2 mM and at temperatures /= 4 mM. In the presence of Ca2+, such that [Ca2+] + [Mg2+] = 15 mM, the observed rate constants of both transients are constant when 4 mM </= [Mg2+] </= 7 mM but double between 7 and 11 mM Mg2+. At pH 6.5 when 4 mM </= [Mg2+] </= 7 mM in the absence of Ca2+, there is also evidence for a third transient with a Mg2+-dependent observed rate constant that is intermediate between the observed rate constants of docking and transesterification. Thus these experiments reveal several separable, local folding transitions that are dependent on Mg2+ in a very cooperative manner and are important for function. When pdG is substituted for prG, no transesterification is observed, and fluorescence quenching is observed for 1-15 mM Mg2+. The switch from fluorescence enhancements with prG to quenching with pdG suggests the 2' OH of prG is important for proper positioning of substrate in the catalytic site.