Explanation by the double-metal-ion mechanism of catalysis for the differential metal ion effects on the cleavage rates of 5′-oxy and 5′-thio substrates by a hammerhead ribozyme

Abstract

In a previous examination using natural all-RNA substrates that contained either a 5-oxy or 5-thio leaving group at the cleavage site, we demonstrated that (i) the attack by the 2-oxygen at C17 on the phosphorus atom is the rate-limiting step only for the substrate that contains a 5-thio group (R11S) and (ii) the departure of the 5 leaving group is the rate-limiting step for the natural all-RNA substrate (R11O) in both nonenzymatic and hammerhead ribozyme-catalyzed reactions; the energy diagrams for these reactions were provided in our previous publication. In this report we found that the rate of cleavage of R11O by a hammerhead ribozyme was enhanced 14-fold when Mg 2؉ ions were replaced by Mn 2؉ ions, whereas the rate of cleavage of R11S was enhanced only 2.2-fold when Mg 2؉ ions were replaced by Mn 2؉ ions. This result appears to be exactly the opposite of that predicted from the direct coordination of the metal ion with the leaving 5-oxygen, because a switch in metal ion specificity was not observed with the 5-thio substrate. However, our quantitative analyses based on the previously provided energy diagram indicate that this result is in accord with the double-metal-ion mechanism of catalysis.Among various catalytic RNAs, the hammerhead-type ribozyme is the smallest and best understood as far as the relationship between structure and function is concerned. Naturally occurring hammerhead ribozymes can be found in some RNA viruses, and they act ''in cis'' during viral replication by the rolling circle mechanism (1-3). The hammerhead ribozyme has been engineered in such a way that it can act ''in trans'' against other RNA molecules (4, 5). The trans-acting hammerhead ribozyme developed by Haseloff and Gerlach (5) consists of an antisense section (stems I and III) and a catalytic domain with a flanking stem II and loop section (Fig. 1a). Because of the small size of hammerhead ribozymes, they are well suited for mechanistic studies, being good representatives of catalytic RNAs. Recently, hammerhead ribozymes were crystallized and their structures were analyzed in detail by two independent groups (6-9). The global three-dimensional structures of the two crystallized ribozymes were nearly identical: one domain of the conserved core, which consists of the sequence C 3 U and is located next to stem I, makes a sharp turn identical to the uridine turn in tRNAs (10). As a result, stem II and stem III are aligned almost colinearly through pseudocontinuous, long A-type helices.It is now well established that ribozymes are metalloenzymes (2,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Although x-ray analysis of a hammerhead ribozyme identified one potential catalytic metal ion (7-9), the exact number of metal ions required for catalysis remains obscure, because the newly captured conformational intermediate appears to demand further conformational change for the following in-line attack (9). Direct evidence that a Mg 2ϩ ion acts as a Lewis acid by coordinating directly to the leaving ...

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

“…Several lines of evidence are consistent with a double-metal-ion mechanism of catalysis ( Fig. 1A), in which two Mg 2 ions are coordinated directly with the attacking and the leaving oxygens (Taira et al 1990;Steitz & Steitz 1993;Uchimaru et al 1993;Uebayashi et al 1994;Sawata et al 1995;Kumar et al 1996;Pontius et al 1997;Zhou et al 1997;Lott et al 1998;Zhou & Taira 1998;Yoshinari & Taira 2000).…”

“…The two Mg 2 ions were considered to be the catalytic metal ions at the cleavage site ( Fig. 1A) that are directly coordinated with the attacking 2 H -oxygen and the leaving 5 H -oxygen (Lott et al 1998), as proposed in the double-metal-ion mechanism of catalysis (Taira et al 1990;Steitz & Steitz 1993;Uchimaru et al 1993;Uebayashi et al 1994;Sawata et al 1995;Kumar et al 1996;Pontius et al 1997;Zhou et al 1997;Lott et al 1998;Zhou & Taira 1998). In order to exclude the possibility that one of the metal ions that contributed to the observed bell-shaped curve might have been the conserved P9 metal ion, located between the pro-Rp oxygen of the phosphate moiety of A 9 and N7 of G 10.1 , we monitored the rate of cleavage of S13 as a function of the concentration of La 3 ions in the presence of 8 mM Mg 2 ions (Fig.…”

“…Studies of solvent isotope effects (Sawata et al 1995) and a kinetic analysis of a modi®ed substrate with a 5 Hthio-leaving group at the cleavage site (Zhou et al 1997) lent strong support to the double-metal-ion mechanism of catalysis. Further evidence was provided by von Hippel's group (Pontius et al 1997;Lott et al 1998).…”

“…Base catalysis mediated by Mg 2 -hydroxide was proposed on the basis of pro®les of pH vs. rate of various metal ion-catalysed reactions of hammerhead ribozymes (Dahm et al 1993). The number of Mg 2 ions that are directly involved in catalysis by hammerhead ribozymes remains to be clari®ed (Taira et al 1990;Pyle 1993;Steitz & Steitz 1993;Uchimaru et al 1993;Yarus 1993;Uebayashi et al 1994;Sawata et al 1995;Kumar et al 1996;Hermann et al 1997;Pontius et al 1997;Zhou et al 1997;Lott et al 1998;Murray et al 1998a, b;Zhou & Taira 1998;Hansen et al 1999;Morrissey et al 1999;Yoshinari & Taira 2000). A general double-metal-ion mechanism Y Nakamatsu et al 604 Genes to Cells (2000) 5, 603±612 q Blackwell Science Limited Figure 1 The complex of the hammerhead ribozyme R34 and its substrate S13, together with potential metal-ion-binding sites in the complex.…”

Explanation by a putative triester‐like mechanism for the thio effects and Mn2+ rescues in reactions catalyzed by a hammerhead ribozyme

“…It is now well established that metal ions are absolutely required in hammerhead ribozyme reactions, as structural folding agents [1–3]and also as catalytic cofactors [4–11]. Thus, hammerhead ribozymes have been recognized as metalloenzymes [1–15]. Recent X‐ray structure by Scott's and Uhlenbeck's group identified a metal‐ion binding site at position 9 (Fig.…”