Probing the Role of Metal Ions in RNA Catalysis:  Kinetic and Thermodynamic Characterization of a Metal Ion Interaction with the 2‘-Moiety of the Guanosine Nucleophile in the Tetrahymena Group I Ribozyme

Abstract

Deciphering the role of individual metal ions in RNA catalysis is a tremendous challenge, as numerous metal ions coat the charged backbone of a folded RNA. Metal ion specificity switch experiments combined with quantitative analysis may provide a powerful tool for probing specific metal ion-RNA interactions and for delineating the role of individual metal ions among the sea of metal ions bound to RNA. We show herein that Mn 2+ rescues the deleterious effect of replacing the 2′-OH of the guanosine nucleophile (G) by -NH 2 (G NH 2 ) in the reaction catalyzed by the Tetrahymena group I ribozyme (E), and the Mn 2+ concentration dependence suggests that a single metal ion is responsible for rescue. This provides strong evidence for a metal ion interaction with the 2′-moiety of G in this ribozyme (referred to as M C ), confirming and extending previous results in a bacteriophage group I intron [Sjögren, A.-S., Pettersson, E., Sjöberg, B.-M., and Strömberg, R. (1997) Nucleic Acids Res. 25, 648-654]. Toward understanding the >10 6 -fold catalytic contribution of the 2′-OH of G, we have determined the individual reaction steps affected by M C and quantitated these effects. Mn C 2+ has only a small effect on binding of G NH 2 to the free ribozyme or ribozyme‚oligonucleotide complexes that lack the reactive phosphoryl group. In contrast, Mn C 2+ increases the binding of G NH 2 to the ribozyme‚oligonucleotide substrate (E‚S) complex 20-fold and increases the binding of S to the E‚G NH 2 complex by the same amount. These and other observations suggest that M C plays an integral role in the coupled binding of the oligonucleotide substrate and the guanosine nucleophile. This metal ion may be used to align the nucleophile within the active site, thereby facilitating the reaction. Alternatively or in addition, M C may act in concert with an additional metal ion to coordinate and activate the 3′-OH of G. Finally, these experiments have also allowed us to probe the properties of this metal ion site and isolate the energetic effects of the interaction of this specific metal ion with the 2′-moiety of G.Divalent metal ions are essential for the structure and function of RNA. Numerous metal ions typically coat an RNA to neutralize the backbone charges and stabilize tertiary contacts, facilitating the folding of RNA into specific threedimensional structures (e.g., 2-19 and references cited therein). In addition to these structural roles, specific active site metal ions also directly participate in the chemical transformations catalyzed by many RNA enzymes (e.g., 20-26). However, the sea of metal ions bound to RNA presents an enormous challenge to deciphering the role of individual metal ions in RNA catalysis.Specific metal ion ligands have been identified by metal ion specificity switch experiments (20-32). These experiments exploit the preference of nitrogen and sulfur ligands to interact more strongly with soft metal ions, such as Mn 2+ , than with hard metal ions, such as Mg 2+ . The identification of metal ion ligands pro...

Dissection of a metal-ion-mediated conformational change in Tetrahymena ribozyme catalysis

“…The model in Figure 2 predicts that at low temperature, the interaction of M C with the 2 ' moiety of G will be made even in the absence of bound S+ To test this model, we determined the effect of replacing the Mg 2ϩ ion at site C with Mn 2ϩ on the binding of G N to the free ribozyme and to the E•S complex at 4 8C+ In principle, the specific Mn C 2ϩ •G N interaction needs to be isolated by comparing the effect of replacing Mg C 2ϩ with Mn C 2ϩ on the binding of G N relative to G (Shan & Herschlag, 1999;Shan et al+, 1999a)+ This analysis can be simplified, however, as previous work showed that the binding and reactivity of G and S are the same with Mg 2ϩ or Mn 2ϩ bound at site C (Shan & Herschlag, 1999; data not shown)+ Thus, this aspect of the analysis is not explicitly shown+ Previous work has also established conditions under which Mn 2ϩ replaces Mg 2ϩ at site C, showing that Mn 2ϩ binds 50-fold stronger than Mg 2ϩ , with apparent dissociation constants of 0+28 and 0+21 mM for free E and the E•S complex, respectively, in the presence of 10 mM Mg 2ϩ (30 8C;Shan & Herschlag, 1999;Shan et al+, 1999b)+ 3 Experiments analogous to those carried out at 30 8C showed that at 4 8C, Mn 2ϩ binds to site C in free E and the E•S complex with apparent dissociation constants of 0+28 and 0+20 mM (in 10 mM Mg 2ϩ ), respectively, the same as the Mn C 2ϩ affinity previously observed at 30 8C (data not shown; see footnote 3)+ 4 The Mn C 2ϩ affinity for the E•G N and E•S•G N complexes are stronger than those for free E and the E•S complex; these stronger Mn C 2ϩ affinities presumably arise from the strong interaction of Mn 2ϩ with the 2 ' -amino group of G N in the complexes at 4 8C+ 5…”

“…To determine whether the metal ion at site C interacts with the 2 ' moiety of G in the E•S•G N complex, the binding of G N to the E•S complex was probed in presteady-state kinetic experiments using the wild-type oligonucleotide substrate, rSA 5 (Table 1), with Mg 2ϩ or Mn 2ϩ bound at site C+ To ensure that the chemical step is rate limiting, the substrate Ϫ1d,rSA 5 (Table 1) was also used; replacement of the 2 ' -OH of U(Ϫ1) with 2 ' -H slows the rate of the chemical step 10 3 -fold, without 3 Ten millimolar Mg 2ϩ was present to ensure proper folding of the ribozyme and to minimize nonspecific binding of Mn 2ϩ to other metal ion sites (Shan & Herschlag, 1999)+ Because a Mg 2ϩ ion is bound at site C at Mg 2ϩ concentrations above 2 mM, the experimentally determined Mn 2ϩ affinities are apparent affinities, representing the exchange of Mn C 2ϩ for Mg C 2ϩ in the presence of 10 mM Mg 2ϩ (Shan & Herschlag, 1999)+ 4 The similar apparent Mn C 2ϩ affinities for E and the E•S complex in the presence of Mg 2ϩ are consistent with the similar Mn 2ϩ and Mg 2ϩ affinities for oxygen ligands (Martell & Smith, 1976;Shan & Herschlag, 1999), such as the nonbridging oxygen of the reactive phosphoryl group of S, as these are ligand exchange reactions with water molecules+ Thus, the presence of S would not be expected to affect the affinity of Mn 2ϩ relative to Mg 2ϩ at site C+ 5 These conclusions are derived from the following observations and analyses+ Replacing Mg C 2ϩ with Mn C 2ϩ increases the G N affinity for free E 20-fold at 4 8C (Table 2)+ Mn C 2ϩ is therefore predicted to bind to the E•G N complex 20-fold stronger than to free E, according to the thermodynamic cycle of equation 2:…”

“…Thermodynamic framework for the conformational change of the ribozyme mediated by interactions of the metal ion at site C with the substrates at 30 8C (A) and 4 8C (B)+ The model is depicted for the Mn C 2ϩ •G N pair, and was constructed using the observed Mn C 2ϩ effects on the binding of S and G N to the ribozyme and ribozyme•substrate complexes at the two different temperatures, as described in the Results+ K a S and K a G N denote the equilibrium association constants of S and G N , respectively, in the unaligned state (top planes in blue), K a S ' and K a G N ' denote the equilibrium association constants of S and G N , respectively, in the aligned state (bottom planes in red), and K ⌬ is the equilibrium constant for going from the unaligned to the aligned conformation+ (Figure continues on facing Shan & Herschlag, 1999), suggesting that the Mn 2ϩ at site C interacts with the 2 ' -amino group of G N in the presence of bound S, that is, in the E•S•G N complex+…”

“…in which an exogenous guanosine nucleophile (G) cleaves a specific phosphodiester bond of an oligonucleotide substrate (S)+ The well-characterized catalytic activity of this ribozyme provides a sensitive assay that can be used to probe its structure and conformation (Herschlag & Cech, 1990)+ Extensive mechanistic work and mutational analyses have provided a detailed model for active site interactions that are crucial for catalysis (Fig+ 1;Piccirilli et al+, 1993;Weinstein et al+, 1997;Strobel & Ortoleva-Donnelly, 1999;Shan & Herschlag, 1999;Shan et al+, 1999aShan et al+, , 2001Yoshida et al+, 2000, and refs therein)+ The tools developed in these studies Abbreviations: E denotes the Tetrahymena thermophila L-21 ScaI ribozyme; S denotes the oligonucleotide substrate with the sequence CCCUCUA 5 , without specification of modifications at the 2 ' -hydroxyl group (the individual oligonucleotide substrates used in this work are listed in Table 1); P denotes the oligonucleotide product with the sequence CCCUCU; G denotes the guanosine nucleophile, and G N denotes the guanosine analog in which the 2 ' -hydroxyl of G is replaced by a 2 ' -amino group+ M C refers to the metal ion at site C ( There is evidence for a conformational change within the active site upon binding of the oligonucleotide substrate and the guanosine nucleophile+ The presence of bound S at the active site increases the affinity of the ribozyme for the guanosine nucleophile, and vice versa (McConnell et al+, 1993;Bevilacqua et al+, 1994)+ Further, this coupling effect is dependent on temperature: S and G enhance the binding of one another only above 10 8C, whereas at lower temperature, the guanosine nucleophile can bind as strongly to the free ribozyme as to the E•S complex even when S is not bound (McConnell & Cech, 1995)+ This strong substrate binding in the absence of the other substrate at low temperature suggested that coupling does not arise from a direct interaction between the two substrates+ Rather, binding of S or G provides the energy for the change of the ribozyme to a conformation in which active site groups are better aligned to interact with the other substrate+ Below 10 8C, this conformation is apparently adopted by the ribozyme without the free energy from substrate binding, thereby allowing strong binding of S or G even in the absence of the other substrate (McConnell & Cech, 1995)+ What are the molecular interactions involved in this conformational change? The first clue came from the observation that the presence of bound oligonucleotide product (P) does not increase the affinity for G, even though P is more stably bound at the ribozyme active site than S; this indicates that the reactive phosphoryl group of S is required (McConnell et al+, 1993)+ The second clue came from the observation that coupling is lost when the 2 ' -OH of G is replaced by 2 ' -H, indicating that the 2 ' -OH of G is also involved (Li & Turner, 1997)+ Recently, a metal ion was identified that appears to coordinate both the p...…”

“…The first clue came from the observation that the presence of bound oligonucleotide product (P) does not increase the affinity for G, even though P is more stably bound at the ribozyme active site than S; this indicates that the reactive phosphoryl group of S is required (McConnell et al+, 1993)+ The second clue came from the observation that coupling is lost when the 2 ' -OH of G is replaced by 2 ' -H, indicating that the 2 ' -OH of G is also involved (Li & Turner, 1997)+ Recently, a metal ion was identified that appears to coordinate both the pro-S P oxygen of the reactive phosphoryl group and the 2 ' -OH of G (Fig+ 1, M C ; Shan et al+, 2001)+ These results suggest that the conformational change upon substrate binding may be mediated by this bridging metal ion+ Interactions with the 2 ' -OH of G could alter the position of M C so that this metal ion becomes better aligned for interaction with the pro-S P oxygen of the reactive phosphoryl group, and vice versa+ Thermodynamic evidence for an integral role of M C in mediating the coupling between S and G came from mechanistic analyses of the effect of this metal ion on individual reaction steps (Shan & Herschlag, 1999)+ When the 2 ' -OH of G is replaced by 2 ' -NH 2 (G N ) and metal site C is occupied by Mg 2ϩ , the coupling between the oligonucleotide substrate and the nucleophile is lost, presumably because of the weaker interaction of Mg 2ϩ with the 2 ' -NH 2 than the 2 ' -OH group+ When the Mg 2ϩ ion at site C is replaced by Mn 2ϩ , coupling is restored, presumably because Mn 2ϩ can make a stronger interaction with the 2 ' -amino group than Mg 2ϩ + As expected, the interaction of Mn C 2ϩ with the 2 ' -NH 2 of G N requires the presence of the reactive phosphoryl group at 30 8C (Shan & Herschlag, 1999), analogous to the requirement of the reactive phosphoryl group for the coupling between S and G at this temperature (McConnell et al+, 1993)+ In this work, we further tested the role of M C in the coupling between S and G by probing the interaction of M C with the 2 ' -moiety of G at 4 8C; at this temperature, the guanosine nucleophile binds strongly to E even in the absence of S, most likely because low temperature favors the conformation of the ribozyme in which the substrates are already positioned to interact with M C + Consistent with predictions from this temperature-dependent conformational change, we found that interaction of the metal ion at site C with the 2 ' -functional group of G can be made even in the absence of bound S at 4 8C+ These results provide additional support for the proposed conformational change and for a crucial role of M C in this change+ Importantly, analysis of the data from this and the previous studies allows construction of a quantitative two-state model for this ribozyme conformational change+ FIGURE 1. Model for catalytic interactions at the Tetrahymena ribozyme active site+ The transition state of the reaction is shown, with the dashed lines (-) depicting the partial bonds from the reactive phosphorus to the leaving group and the incoming nucleophile, and d-depicting the partial negative charges on the leaving group and the nucleophile+ The dots (•) depict interactions of metal ions with their ligands, and thick dashed lines depict hydrogen bonding interactions+ M A , M B , and M C are the three previously identified catalytic metal ions at this RNA active site (Shan et al+, 1999a)+ M A coordinates the 3 ' -bridging oxygen of S and the pro-S P oxygen of the reactive phosphoryl group (Piccirilli et al+, 1993;Shan et al+, 2001), M B coordinates the 3 ' -oxygen...…”