Inhibition of the Hammerhead Ribozyme Cleavage Reaction by Site-Specific Binding of Tb(III)

Abstract

Terbium(III) [ Tb(III)] was shown to inhibit the hammerhead ribozyme by competing with a single magnesium(II) ion. X-ray crystallography revealed that the Tb(III) ion binds to a site adjacent to an essential guanosine in the catalytic core of the ribozyme, approximately 10 angstroms from the cleavage site. Synthetic modifications near this binding site yielded an RNA substrate that was resistant to Tb(III) binding and capable of being cleaved, even in the presence of up to 20 micromolar Tb(III). It is suggested that the magnesium(II) ion thought to bind at this site may act as a switch, affecting the conformational changes required to achieve the transition state.RNA enzymes require divalent metal ions for activity, either to promote folding or for direct participation in catalysis. The hammerhead ribozyme (Fig. 1A), a self-cleaving RNA found naturally in plant viroids and virusoids, is an excellent system in which to study metal ion-RNA interactions because of the extensive structural and mechanistic data available (1-4). Two independent crystal structures of the hammerhead ribozyme have revealed divalent metal ions binding to six different sites on the molecule (5-7). Biochemical methods available to evaluate the role of these metal ions in ribozyme function are limited. The most common approach is to introduce a phosphorothioate modification into the RNA and to examine its effect on the metal specificity of the catalytic reaction (8-13). We present an approach to studying metal binding to ribozymes based on the observation that ions that compete efficiently for critical Mg-binding sites can thereby inhibit catalysis. The powerful enzymatic and spectroscopic tools originally developed for use with protein metalloenzymes can then be applied to RNA systems such as the hammerhead ribozyme.Interactions between lanthanide ions and RNA molecules have been studied (14,15). (Fig. 1B). This signal was absent from control samples lacking either RNA or Tb(III). These data indicate that the Tb ion binds to the RNA, resulting in energy transfer from the RNA to the lanthanide ion. We therefore investigated the effects of Tb(III) binding on the hammerhead-catalyzed reaction, a site-specific cleavage of a phosphodiester bond to form 2Ј,3Ј-cyclic phosphate and 5Ј-hydroxyl termini.Terbium(III) proved an efficient inhibitor of hammerhead cleavage (18). For example, Tb(III) inhibited the HH8 single-turnover cleavage reaction with an apparent inhibition constant (K i,app ) of 2.0 Ϯ 0.3 M at 25 mM Mg(II) (Fig. 2A), and similar values were obtained under multiple-turnover conditions. Two other well-characterized hammerheads, HH16 (19) and HH␣1 (20), showed K i,app values for Tb(III) of 1.1 Ϯ 0.4 and 0.57 Ϯ 0.08 M, respectively, at 10 mM Mg(II) (21). Because all three ribozymes were inhibited similarly by Tb(III) despite sequence differences in the peripheral base-paired regions and loops, the data indicate that the binding event that underlies inhibition results from the interaction of Tb(III) with a site in the conserved catalytic...

Significant activity of a modified ribozyme with N7‐deazaguanine at G10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

“…In some cases, another metal-binding site has been found close to G 5 and replacement here of Mg 2 by Tb 3 inhibited the cleavage reaction (Feig et al 1998). However, in the case of the metal-binding site near G 5 , the introduction of a sterically bulky group prevented the Tb 3 ionmediated inhibition, suggesting that the Mg 2 ion at this site is bound only to the ground-state conformation and is released before the transition state is achieved (Feig et al 1998). Therefore, according to crystallographic and biochemical studies, the metal ion that is essential for catalysis appears to be the P9 metal ion [in addition to the metal ion(s) at the cleavage site], despite its considerable distance (<20 A Ê ) from the reactive phosphoryl group (Pley et al 1994;Scott et al 1995).…”

Metal ion cooperativity in ribozyme cleavage of RNA

“…Cleavage reactions were performed at 37°C as described in detail elsewhere (15) in our standard reaction buffer: 50 mM Tris-HCl (pH 7.2), 5% (wt͞vol) polyethylene glycol 6000, 100 mM NH 4 Cl, and 40 mM total concentration of different divalent metal ions as indicated. The chloride salt of the various Me 2ϩ was used except for Ba 2ϩ , for which we used barium acetate.…”

“…The role of the divalent metal ions can be structural and͞or catalytic, and in RNA-catalyzed reactions the existence of several categories of divalent metal ion binding sites has been suggested (1)(2)(3)(4)(5). The reaction catalyzed by the ribozyme RNase P RNA, the catalytic subunit of the endoribonuclease RNase P (6), requires divalent metal ions; among the ones studied thus far, Mg 2ϩ promotes cleavage most efficiently.…”

In Vitro Selection of a New Lanthanide-Dependent DNAzyme for Ratiometric Sensing Lanthanides

“…[22][23][24][25][26][27][28] 13 and the hammerhead ribozyme. 37 The first DNAzyme, GR5, was also found to be active with lanthanides alone, although the rate was quite slow (below 0.02 min -1 ). 38 We recently selected a DNAzyme (named Ce13d) using Ce 4+ as the intended target.…”