Studies on Escherichia coli RNase P RNA with Zn²⁺ as the catalytic cofactor

Abstract

We demonstrate, for the first time, catalysis by Escherichia coli ribonuclease P (RNase P) RNA with Zn²⁺ as the sole divalent metal ion cofactor in the presence of ammonium, but not sodium or potassium salts. Hill analysis suggests a role for two or more Zn²⁺ ions in catalysis. Whereas Zn²⁺ destabilizes substrate ground state binding to an extent that precludes reliable K_d determination, $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ and Sr²⁺ in particular, both unable to support catalysis by themselves, promote high-substrate affinity. Zn²⁺ and $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ substantially reduce the fraction of precursor tRNA molecules capable of binding to RNase P RNA. Stimulating and inhibitory effects of Sr²⁺ on the ribozyme reaction with Zn²⁺ as cofactor could be rationalized by a model involving two Sr²⁺ ions (or two classes of Sr²⁺ ions). Both ions improve substrate affinity in a cooperative manner, but one of the two inhibits substrate conversion in a non-competitive mode with respect to the substrate and the Zn²⁺. A single 2′-fluoro modification at nt −1 of the substrate substantially weakened the inhibitory effect of Sr²⁺. Our results demonstrate that the studies on RNase P RNA with metal cofactors other than Mg²⁺ entail complex effects on structural equilibria of ribozyme and substrate RNAs as well as E·S formation apart from the catalytic performance.

INTRODUCTION

The ribonucleoprotein enzyme ribonuclease P (RNase P) is an endonuclease that generates the mature 5′ ends of tRNAs in all three domains of life (Archaea, Bacteria and Eukarya) as well as in the mitochondria and the chloroplasts (1–3). Bacterial RNase P enzymes are composed of a catalytic RNA subunit, ∼400 nt in length, and a single small protein of typically 120 amino acids (4,5). Studies with RNase P RNA from Escherichia coli (structural RNase P RNA subtype A) and Bacillus subtilis (subtype B) have implied a specific role for two or more metal ions in substrate binding and cleavage chemistry (6–13). Mg²⁺ and Mn²⁺ efficiently support the precursor tRNA (ptRNA) processing reaction catalyzed by bacterial RNase P RNAs (14), which generates 3′-OH and 5′-phosphate termini. For E.coli RNase P RNA (referred to as M1 RNA further), processing under standard assay conditions was reported to be essentially abolished when Mg²⁺ or Mn²⁺ is replaced with earth alkaline metals such as Sr²⁺, transition metal ions, such as Zn²⁺, Co²⁺ and Ni²⁺, or $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ as a potential mimic of hexaaquo Mg²⁺ (11,15). Ca²⁺, as an exception, supported the reaction, although inefficiently (11,14). Different results were obtained with a ptRNA carrying an Rp-phosphorothioate modification at the RNase P cleavage site. This substrate was cleaved quite efficiently by M1 RNA when Mg²⁺ was replaced with Cd²⁺, conditions under which cleavage of the unmodified ptRNA was hardly detectable (7). This result suggested that the failure of transition metal ions such as Cd²⁺ to support cleavage of unmodified ptRNA by M1 RNA is due to their inability to interact properly with the phosphate pro-Rp oxygen at the scissile phosphodiester in the transition state. The situation was found to be somewhat different in the reaction catalyzed by B.subtilis RNase P RNA. This ribozyme required a second metal ion, such as Ca²⁺, in addition to Cd²⁺ for processing a ptRNA with a single Rp-phosphorothioate modification at the cleavage site. Here, Ca²⁺ was inferred to be essential for productive enzyme–substrate complex formation (8), suggesting that there are differential roles for metal ions in RNase P RNA-catalyzed reactions. Synergistic effects of metal ion combinations were also observed for the reaction catalyzed by E.coli M1 RNA, although their molecular basis has been poorly understood. While the ribozyme failed to cleave the substrate in the presence of Ba²⁺, Sr²⁺, Zn²⁺ or $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ alone, some cleavage activity was restored with the combinations Zn²⁺/Sr²⁺, Zn²⁺/Ba²⁺ or ${\text{Zn}}^{2+}/\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ (11). Catalysis by M1 RNA with Sr²⁺ or Ba²⁺ alone has only been observed with low efficiency under very specialized conditions [at pH≫7 and in the presence of ethanol; (16)]. From the Pb²⁺-induced hydrolysis patterns of M1 RNA generated in the presence of different divalent metal ions and $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ (17), it was concluded that the M1 RNA conformation is very similar in the presence of Mg²⁺, Mn²⁺, Ca²⁺, Sr²⁺, Ba²⁺ and apparently also $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$, whereas transition metals, such as Zn²⁺ and particularly Cd²⁺, Co²⁺, Cu²⁺ and Ni²⁺, induce changes of the native M1 RNA conformation.

To obtain a deeper insight into how different metal ions modulate this ribozyme system, we have investigated the effects of Zn²⁺, Sr²⁺ and $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ on different aspects of processing by M1 RNA: catalysis and its inhibition by Sr²⁺ in particular, enzyme–substrate affinity and changes in the fraction of substrate able to bind to the enzyme. Catalysis was analyzed under conditions of E≫S, and all assays included a relatively high monovalent salt concentration (1 M NH₄OAc) to focus on the roles of metal ions which cannot be fulfilled by monovalent cations. To further characterize the metal ion interaction in vicinity of the 2′-OH group at nt −1 of the substrate, we have tested cleavage by M1 RNA in the presence of Zn²⁺/Sr²⁺ with ptRNA substrates carrying a single 2′-amino (2′-N), 2′-fluoro (2′-F) or 2′-deoxy (2′-H) substitution at nt −1. Such 2′-ribose modifications at the RNase P cleavage site were previously reported to affect the binding of catalytically important Mg²⁺ and to substantially reduce the rate of catalysis by M1 RNA (6,18–20). Recent NMR experiments have provided further evidence that the metal ion coordinated with the help of the 2′-OH group at nt −1 is actually ‘pre-bound’ to ptRNA before complexation with RNase P RNA (21).

MATERIALS AND METHODS

RNA synthesis and labeling

Chemical and enzymatic RNA synthesis, purification of RNA and assembly of ptRNA variants with single-site modifications have been described recently (20).

Kinetics

Processing assays were performed at 37°C under single turnover conditions (5 μM M1 RNA, <1 nM ptRNA, 1 M NH₄OAc, 50 mM MES for the pH range of 5–6 and PIPES for pH 6–7; metal ion concentration and pH at 37°C as indicated) as described previously (22). Aliquots withdrawn from the enzyme–substrate mixtures were desalted by ethanol precipitation in the presence of 20 μg glycogen before analysis by 20% PAGE/8 M urea. Data analysis and calculation of single turnover rates of cleavage (k_obs) were performed as described previously (22).

The dependence of k_obs on [Sr²⁺] in Figure 7A and B showed a stimulatory and inhibitory phase. These primary data were replotted as k_obs in the absence of Sr²⁺ divided by k_obs at individual Sr²⁺ concentrations (termed v₀/v_i) over [Sr²⁺] (Figure 7C and D). These secondary plots were fit to the equation v₀/v_i = (K_S + [E](1 + (1/αK_I(a) + 1/βK_I)[I] + [I]²/αβK_I(a) K_I))/((K_S + [E])(1 + [I]/αK_I(a))), derived from a model (Figure 8A) involving two Sr²⁺ ions (or two classes of Sr²⁺ ions): both improve substrate affinity in a cooperative manner, but one of the two inhibits substrate conversion in a non-competitive mode with respect to the substrate. The equation was derived as follows [E, M1 RNA; S, ptRNA substrate; I(a), Sr²⁺ with ‘activating’ effect owing to a reduction in K_S, expressed as α · K_S; I, inhibitory Sr²⁺ which also reduces K_S, expressed as β · K_S].

Figure 7.

Sensitivity of processing rates as a function of [Sr²⁺] to differences in [E]: (A) all-ribose ptRNA^Gly (2′-OH); (B) 2′-F-ptRNA^Gly. (C and D) Secondary v₀/v_i plots of the data (v₀ = k_obs in the absence of Sr²⁺; v_i = k_obs at the respective Sr²⁺ concentration) from (A) and (B). Data fitting was best with the model depicted in Figure 8A, using Equation 8 (see Materials and Methods and Figure 8): v₀/v_i = (K_S + [E] (1 + (1/αK_I(a) + 1/βK_I) [I] + [I]²/αβK_I(a) K_I))/((K_S + [E]) (1 + [I]/αK_I(a))). Curve fits yielded the following values for αK_I(a) and βK_I: 1.46 ± 0.46 mM and 0.72 ± 0.025 mM [(C), 1.4 μM M1 RNA, 2′-OH], 1.49 ± 0.45 mM and 0.72 ± 0.005 mM [(C) 5 μM M1 RNA, 2′-OH], 3.5 ± 0.4 and 9.1 ± 2.1 mM [(D) 1.4 μM M1 RNA, 2′-F] and 2.2 ± 0.2 and 11 ± 0.8 mM [(D) 5 μM M1 RNA, 2′-F]; estimates for K_S were 50 and 120 μM in (C) and (D), respectively.

Figure 8.

(A) Equilibria for the M1 RNA-catalyzed processing reaction in the presence of constant [Zn²⁺] as the catalytic cofactor and varying concentrations of [Sr²⁺] that activate the reaction at low but inhibit at higher concentrations. The model involves two Sr²⁺ ions (or two classes of Sr²⁺ ions) that both improve substrate affinity in a cooperative manner (see Figure 3B), but one of the two inhibiting substrate conversion non-competitively with respect to the substrate. Other models, for example, assuming the involvement of two ‘activating’ and one inhibitory Sr²⁺ ion or predicting that E·S·I and E·S·I(a)·I complexes retain residual reactivity, failed to give satisfactory curve fits of the data in Figure 7C and D. I(a) = Sr²⁺ ion that activates processing by decreasing K_S to αK_S; I = inhibitory Sr²⁺ ion that inhibits cleavage chemistry but also decreases K_S to βK_S. (B) Dixon plot of the M1 RNA-catalyzed ptRNA cleavage rate at 37°C as a function of [Sr²⁺] in the presence of four different fixed Zn²⁺ concentrations and 5 μM M1 RNA, <1 nM ptRNA, 50 mM PIPES, pH 6.65 and 1 M NH₄OAc; open circles, 8.1 mM Zn²⁺; filled circles, 10 mM Zn²⁺; open squares, 15 mM Zn²⁺; and filled triangles, 20 mM Zn²⁺. All four datasets fit to straight lines intersecting on the x-axis, indicating that Sr²⁺ acts as a non-competitive inhibitor with respect to Zn²⁺. The point of intersection on the x-axis yields a K_I-value of ∼5 mM.

The velocity dependence equation is:

$$v_{\text{i}}=k_{\text{chem}}·\left(\left[\text{ES}\right]+\left[\text{ESI}\left(\text{a}\right)\right]\right).$$ 1

Dividing both sides of the velocity dependence Equation 1 by [S]_t:

$$\frac{v_{\text{i}}}{{\left[\text{S}\right]}_t}=\frac{k_{\text{chem}}·\left(\left[\text{ES}\right]+\left[\text{ESI}\left(\text{a}\right)\right]\right)}{{\left[\text{S}\right]}_t}.$$ 2

$${\left[\text{S}\right]}_t=\left[\text{S}\right]+\left[\text{ES}\right]+\left[\text{ESI}\right]+\left[\text{ESI}\left(\text{a}\right)\right]+\left[\text{ESI}\left(\text{a}\right)\text{I}\right].$$ 3

Replacing [S]_t in the right-hand side of Equation 2:

$$\frac{v_{\text{i}}}{{\left[\text{S}\right]}_t}=\frac{k_{\text{chem}}·\left(\left[\text{ES}\right]+\left[\text{ESI}\left(\text{a}\right)\right]\right)}{\left[\text{S}\right]+\left[\text{ES}\right]+\left[\text{ESI}\right]+\left[\text{ESI}\left(\text{a}\right)\right]+\left[\text{ESI}\left(\text{a}\right)\text{I}\right]}.$$ 4

Expressing the concentration of each species in terms of [E]:

$$\frac{v_{\text{i}}}{{\left[\text{S}\right]}_t}=\frac{k_{\text{chem}}·\left(\frac{\left[\text{S}\right]·\left[\text{E}\right]}{K_{\text{S}}}+\frac{\left[\text{S}\right]·\left[\text{E}\right]·\left[\text{I}\right]}{\alpha ·K_{\text{S}}·K_{\text{I}\left(\text{a}\right)}}\right)}{\left[\text{S}\right]+\frac{\left[\text{S}\right]·\left[\text{E}\right]}{K_{\text{S}}}+\frac{\left[\text{S}\right]·\left[\text{E}\right]·\left[\text{I}\right]}{\beta ·K_{\text{S}}·K_{\text{I}}}+\frac{\left[\text{S}\right]·\left[\text{E}\right]·\left[\text{I}\right]}{\alpha ·K_{\text{S}}·K_{\text{I}\left(\text{a}\right)}}+\frac{\left[\text{S}\right]·\left[\text{E}\right]·{\left[\text{I}\right]}^2}{\alpha ·\beta ·K_{\text{S}}·K_{\text{I}\left(\text{a}\right)}·K_{\text{I}}}}.$$ 5

Multiplying the numerator and the denominator with K_S/[S] and simplifying:

$$\frac{v_{\text{i}}}{{\left[\text{S}\right]}_t}=\frac{k_{\text{chem}}·\left[\text{E}\right]·\left(1+\frac{1}{\alpha ·K_{\text{I}\left(\text{a}\right)}}·\left[\text{I}\right]\right)}{K_{\text{S}}+\left[\text{E}\right]·\left(1+\left(\frac{1}{\beta ·K_{\text{I}}}+\frac{1}{\alpha ·K_{\text{I}\left(\text{a}\right)}}\right)·\left[\text{I}\right]+\frac{1}{\alpha ·\beta ·K_{\text{I}}·K_{\text{I}\left(\text{a}\right)}}·{\left[\text{I}\right]}^2\right)}.$$ 6

When [I] = 0, v_i equals v₀, and Equation 6 simplifies to:

$$\frac{v_0}{{\left[\text{S}\right]}_t}=\frac{k_{\text{chem}}·\left[\text{E}\right]}{K_{\text{S}}+\left[\text{E}\right]}.$$ 7

[S]_t · k_chem is V_max under conditions [E] > [S].

Dividing Equation 7 by Equation 6 and simplifying:

$$\frac{v_0}{{\left[\text{S}\right]}_t}=\frac{K_{\text{S}}+\left[\text{E}\right]·\left(1+\left(\frac{1}{\beta ·K_{\text{I}}}+\frac{1}{\alpha ·K_{\text{I}\left(\text{a}\right)}}\right)·\left[\text{I}\right]+\frac{1}{\alpha ·\beta ·K_{\text{I}}·K_{\text{I}\left(\text{a}\right)}}·{\left[\text{I}\right]}^2\right)}{(K_{\text{S}}+\left[\text{E}\right])·\left(1+\frac{1}{\alpha ·K_{\text{I}\left(\text{a}\right)}}·\left[\text{I}\right]\right)}.$$ 8

Spin column assays

Spin column assays for the determination of equilibrium dissociation constants (K_d) of enzyme–substrate complexes were performed as described previously (8,23) in a buffer containing 50 mM MES, pH 6.0, 1 M NH₄OAc, 0.1% (w/v) SDS, 0.05% (w/v) Nonidet P-40, and indicated concentrations of SrCl₂, Zn(OAc)₂ and/or Co(NH₃)₆Cl₃. For the Hill plot analysis shown in Figure 3, see (9).

Figure 3.

K_d values for ptRNA^Gly binding to M1 RNA determined by the spin column assay (8,23). Assay buffers contained 50 mM MES, pH 6.0, 1 M NH₄OAc and indicated concentrations of Sr²⁺ and/or Zn²⁺. (A) Bar diagram of log K_d dependence on Sr²⁺ and/or Zn²⁺ (or Cd²⁺) concentration as indicated below the diagram. Values above bars represent the proportion of ptRNA^Gly that was able to form a complex with M1 RNA at the theoretical endpoint (M1 RNA saturation), normalized to conditions of 40 mM Sr²⁺. Asterisk above the black bar for conditions of 20 mM Zn²⁺ alone: the experimental endpoint could not be reached due to very weak complex formation, which resulted in high errors for K_d determinations; the K_d of ≥20 μM is therefore only an estimate. Individual values are based on average on four independent experiments; errors are indicated by error bars. (B) Hill plot analysis of K_d dependence on Sr²⁺ concentration. f = (minimal K_d)/(observed K_d); the minimal K_d at saturating Sr²⁺ concentrations was determined as 2 nM.

RESULTS

The well-characterized bacterial ptRNA^Gly used as the substrate for processing by M1 RNA is illustrated in Figure 1, including the variants carrying single-site 2′-ribose modifications at nt −1.

Figure 1.

Secondary structure of the ptRNA^Gly substrate. 2′-Ribose modifications introduced at the canonical RNase P cleavage site (nt −1) are illustrated in the gray-shaded box on the left. The arrow marks the canonical RNase P cleavage site between nucleotides −1 and +1. For further details, see (20).

Cleavage in the presence of Zn²⁺ alone or combinations of Zn²⁺/Sr²⁺ and ${\text{Zn}}^{2+}/\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$

Surprisingly, Zn²⁺ as the only divalent metal ion was able to support ptRNA processing by M1 RNA, but only in the presence of ammonium salts (Figure 2A). The nature of the counter anion (chloride or acetate) was not critical (Figure 2A), although some RNA degradation was observed with chloride salts. Neither significant degradation nor precipitation of ptRNA or M1 RNA occurred under our standard assay conditions (1 M NH₄OAc, 20–80 mM Zn[OAc]₂, pH 6.65; data not shown). Sr²⁺ (Figure 2B) or $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ (data not shown) alone did not support M1 RNA-catalyzed cleavage, in line with the previous observations (11).

Figure 2.

Processing of the all-ribose ptRNA^Gly by E.coli M1 RNA under single turnover conditions at 37°C in the presence of 20 mM Zn²⁺. (A) Dependence on the type of monovalent cation; processing occurred in the presence of ammonium, but not sodium or potassium salts; reaction conditions: 5 μM M1 RNA, <1 nM ptRNA, 50 mM PIPES, pH 6.65, 1 M monovalent salt as indicated, and 20 mM ZnCl₂ or Zn(OAc)₂; control lanes 1, 6, 11, 16 and 21: incubation for 2 h in the absence of M1 RNA; time points were 5 min (lanes 2, 7, 12, 17 and 22), 20 min (lanes 3, 8, 13 and 18), 1 h (lanes 4, 9, 14 and 19) and 2 h (lanes 5, 10, 15 and 20); lanes 21 and 22 (C₁, C₂) are equal to lanes 1 and 2. (B) Assay documenting that no processing occurs in the presence of Sr²⁺ as the sole metal ion; reaction conditions: 5 μM M1 RNA, <1 nM ptRNA, 50 mM PIPES, pH 6.65, 1 M NH₄OAc and 100 mM SrCl₂ (left) or 20 mM ZnCl₂ (right). Control lanes 1 and 6: incubation for 3 h in the absence of M1 RNA; time points were 5 min (lane 7), 20 min (lanes 2 and 8), 40 min (lane 3), 1 h (lanes 4 and 9) and 3 h (lanes 5 and 10). (C and D) Processing rates as a function of increasing concentrations of SrCl₂ (C) or Co(NH₃)₆Cl₃ (D); reaction conditions: 5 μM M1 RNA, <1 nM ptRNA, 50 mM PIPES, pH 6.65, 1 M NH₄OAc and 20 mM Zn(OAc)₂; transition points between the stimulatory and the inhibitory phases of the curves are marked by dashed lines.

We then analyzed single turnover cleavage by M1 RNA (see Materials and Methods) in the presence of Zn²⁺ and increasing concentrations of Sr²⁺ or $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ (Figure 2C and D). The pH 6.65 was chosen to combine substantial substrate turnover with conditions where cleavage chemistry mainly determines the rate of cleavage at saturating enzyme concentrations (7). At constant 20 mM Zn²⁺, both Sr²⁺ and $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ started to stimulate substrate turnover at lower concentrations, followed by an inhibitory phase at higher concentrations (Figure 2C and D). This suggested that the two different Sr²⁺ or $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ ions (or classes of ions) affected the cleavage reaction under these experimental conditions.

${\text{Zn}}^{2+}/{\text{Sr}}^{2+}/\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ dependence of substrate binding to M1 RNA

As a next step, we analyzed the binding of ptRNA^Gly to M1 RNA as a function of Zn²⁺, Sr²⁺ and/or $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$. The following results were obtained by a spin column assay (see Materials and Methods) and are summarized in Table 1. In the absence of any metal ion, a K_d of ∼10 μM was measured under our assay conditions. The K_d increased to ≥20 μM in the presence of 20 mM Zn²⁺, demonstrating that the Zn²⁺ destabilizes the substrate ground state binding. Another transition metal ion, Cd²⁺, supported E·S formation poorly as well (K_d of 2.1 μM), although more efficiently than the Zn²⁺. In contrast to the transition metal ions, Sr²⁺ or $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ support substrate binding, Sr²⁺ more efficiently than $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$. K_d values in the presence of 20 mM Zn²⁺ progressively decreased with increasing Sr²⁺ concentrations (Figure 3A). However, at all tested Sr²⁺ concentrations (5, 10, 20, 40 and 80 mM), the addition of 20 mM Zn²⁺ resulted in a constant 2–3-fold increase in K_d (Figure 3A). Moreover, the proportion of substrate that is able to form a stable complex with the enzyme at the endpoint (i.e. point of enzyme saturation; Figure 3A, numbers above bars) decreased with increasing ratios of [Zn²⁺] to [Sr²⁺], for example, the binding-proficient substrate fraction decreased from 0.92 at 20 mM Zn²⁺/80 mM Sr²⁺ to 0.59 at 20 mM Zn²⁺/5 mM Sr²⁺ (normalized to 1.0 measured at 40 mM Sr²⁺ alone, Figure 3A). Hill analysis of K_d in the presence of varying concentrations of Sr²⁺ and in the absence of Zn²⁺ gave a slope of n_H = 1.8 (Figure 3B), suggesting that at least two Sr²⁺ ions are additionally taken up into the enzyme–substrate complex under the applied conditions.

Table 1.

Influence of Zn²⁺, Sr²⁺ and/or $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ on the binding of ptRNA^Gly to E.coli M1 RNA

Metal ion(s)		Kd (nM)	Average endpoint
Transition Me2+	Other
—	—	10 000 ± 3000	0.66
20 mM Zn2+	—	≥20 000a	n.d.a
20 mM Cd2+	—	2100 ± 400	0.75
—	5 mM Sr2+	240 ± 15	0.93
20 mM Zn2+	5 mM Sr2+	700 ± 300	0.59
—	80 mM Sr2+	4 ± 0.5	0.98
20 mM Zn2+	80 mM Sr2+	9 ± 0.5	0.92
20 mM Zn2+	5 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$	2486 ± 400	0.55
—	20 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$	751 ± 200	0.75
20 mM Zn2+	20 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$	350 ± 120	0.65
—	80 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$	79 ± 35	0.26
20 mM Zn2+	80 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$	53 ± 6	0.28

Spin column assay for the determination of K_d values were performed at pH 6.0 and 1 M NH₄OAc using trace amounts of 5′-endlabeled ptRNA^Gly; individual K_d values are based on three to six independent experiments and were calculated by non-linear regression analysis (program Grafit, Erithacus Software) using the equation: f_c = f_t · [P RNA]_free/(K_d + [P RNA]_free), where f_c = fraction of ptRNA in the complex, and f_t = maximum fraction of ptRNA that is able to bind to P RNA (endpoint). Endpoints in the right column are the theoretical ones obtained by the fitting procedure; however, theoretical and experimentally measured endpoints were generally in good agreement; average endpoints were normalized to that for 40 mM Sr²⁺ (see Figure 3A).

^aK_d and endpoint values (n.d. = not determined) could not be determined with reasonably low errors owing to very low ribozyme–substrate affinity; the K_d of 20 000 nM in the presence of 20 mM Zn²⁺ alone is a lower limit estimate.

$\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ also decreased the K_d values, but the substrate affinity was lower relative to the equal concentrations of Sr²⁺ (Table 1), suggesting that $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ is a rather inefficient substitute for hexahydrated Mg²⁺ in this system. Whereas K_d values in the presence of Sr²⁺ were generally increased by the addition of 20 mM Zn²⁺ (Figure 3A), addition of 20 mM Zn²⁺ to 20 or 80 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ tended to lower K_d to some extent compared with the corresponding $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ alone conditions (Table 1). This suggests that Zn²⁺ and $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ weakly complement each other in promoting E·S complex formation. Remarkably, higher concentrations (e.g. 80 mM) of $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ substantially lowered the proportion of ptRNA capable of complex formation under saturating enzyme concentrations [0.26 for 80 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ versus 0.98 for 80 mM Sr²⁺; Table 1].

Zn²⁺ cooperativity in catalysis by M1 RNA

We observed that M1 RNA is able to catalyze ptRNA processing in the presence of Zn²⁺ as the sole divalent metal ion (Figure 2A). On the other hand, Zn²⁺ failed to promote thermodynamically stable E·S complex formation (Table 1 and Figure 3A), in contrast to other divalent metal ions, such as Mg²⁺ or Mn²⁺, which support both catalysis and E·S formation (14). Thus, processing analyses with Zn²⁺ suggested the potential to use Zn²⁺ as a specific tool to study catalysis apart from E·S complex formation.

To assess the number of catalytic Zn²⁺ ions involved, we analyzed single turnover cleavage of ptRNA^Gly in the presence of increasing Zn²⁺ concentrations, either in the presence of constant 12 mM Sr²⁺ or 20 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$, or without any second metal ion (Figure 4). For the conditions including Sr²⁺ and $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$, enzyme concentration (5 μM) was saturated based on our K_d measurements (Table 1 and Figure 3A). In all cases, reasonable fits to the Hill equation resulted in coefficients of n_H = 2.2 (Figure 4A, at constant 12 mM Sr²⁺), n_H = 2.8 (Figure 4B, Zn²⁺ alone) and n_H = 1.8 [Figure 4C, at constant 20 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$]. These n_H-values in the range 1.8–2.8 support the cooperative involvement of two or more Zn²⁺ ions in catalysis by E.coli M1 RNA. Results from the Zn²⁺-alone reaction, although supporting this Zn²⁺ cooperativity, should yet be interpreted with some caution as the enzyme concentration was subsaturating under these conditions (Table 1); thus, we cannot exclude that, in addition to the rate of the catalytic step, changes in the E·S binding equilibrium upon variation of [Zn²⁺] may also have affected the observed cleavage rates.

Figure 4.

Zn²⁺-dependence of M1 RNA-catalyzed cleavage of all-ribose ptRNA^Gly (Hill analysis). The Zn²⁺-concentration was varied in the range of 0–80 mM: (A) at constant [Sr²⁺] (12 mM) and pH 6.0; (B) in the absence of a second metal ion at pH 6.5; (C) at constant [$\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$] (20 mM) and pH 6.0. Data were analyzed by non-linear regression analysis using the Hill equation v = V_max·[Zn²⁺]ⁿ/(K′_Zn + [Zn²⁺]ⁿ) as described previously (8). The Hill coefficient (n_H) was determined as 2.2 ± 0.03 in (A), 2.8 ± 0.02 in (B) and 1.80 ± 0.04 in (C). For further details, see Materials and Methods.

Processing of substrates with 2′-ribose modifications at nt −1 in the presence of Zn²⁺/Sr²⁺

A model of the RNase P RNA cleavage mechanism [(24); Figure 5, model I] proposes that one Mg²⁺ ion (termed here Mg[B]) simultaneously interacts with the OH⁻ nucleophile (inner-sphere) and the 2′-OH at position −1 of the substrate via an inner-sphere water molecule, and directly coordinates to the pro-Rp phosphate oxygen at the cleavage site. In an alternative model (Figure 5, model II), two metal ions (Mg[A] and Mg[B]) directly coordinate to the pro-Rp oxygen, but Mg[A] instead of Mg[B] interacts with the OH⁻ nucleophile via inner-sphere coordination (7,25). In both models, Mg[B] interacts with the 2′-OH function at nt −1 of the substrate via an inner-sphere water molecule.

Figure 5.

Transition state models for phosphodiester hydrolysis by E.coli M1 RNA. The single 2′-OH at nt −1 (in blue) was replaced with a 2′-deoxy, 2′-amino or 2′-fluoro group in the modified substrates analyzed in Figure 6. Putative, catalytically important Mg²⁺ ions are shown in magenta (Mg [A]) or red (Mg [B]); metal ion site [B] was in the focus of the present study. The first transition state model (model I) for hydrolysis of the scissile phosphodiester connecting nt +1 and −1 is derived from that proposed in (24), according to which the Mg²⁺ ion at the site termed [B] here directly coordinates to the pro-Rp phosphate oxygen and OH⁻ nucleophile (in green), and simultaneously interacts with the 2′-OH at position −1 via an inner-sphere water molecule. According to model II, two Mg²⁺ ions, Mg[A] and Mg[B], directly coordinate to the pro-Rp oxygen (7,25), but Mg[A] instead of Mg[B] interacts with the OH⁻ nucleophile via inner-sphere coordination. Additional metal ion interactions at the pro-Sp oxygen (marked Sp; models I and II) and the 3′-bridging oxygen (model I) are conceivable based on strong inhibition effects caused by sulfur substitutions at these positions (7,24,35). The ribose at nt +1 are drawn in the A-helical C3′-endo and the ribose at position −1 in the C2′-endo conformation based on the results of NMR investigations (36,37).

We addressed the possibility that Sr²⁺ may displace catalytically important Zn²⁺ at the aforementioned metal ion binding site [B], assuming that the binding sites for the two different metal ions overlap to such an extent that their binding is mutually exclusive. We have recently shown that 2′-substitutions at nt −1 of ptRNA decreased cleavage efficiency by M1 RNA in the order 2′-H ≤ 2′-N < 2′-F < 2′-OH under conditions of rate-limiting chemistry (20). Assuming that Sr²⁺ indeed displaces a Zn²⁺ ion that is bound to site [B] involving the 2′-OH at nt −1 of ptRNA (Figure 5), one would expect that these 2′-modifications change the affinities of Zn²⁺ and Sr²⁺ (which largely differ in their electronic properties and the details of their coordination spheres; see Discussion) to different extents. In contrast, the competition profile should be much less affected if Sr²⁺ displaces catalytic Zn²⁺ at a site other than [B], where metal ion coordination is not directly dependent on the 2′-OH at nt −1. In fact, the concentration of Sr²⁺ at the transition point between the stimulatory and the inhibitory phases of the curve was substantially shifted toward higher Sr²⁺ concentrations (20–40 mM versus 3.5 mM, Figure 6A) when the 2′-OH at nt −1 was replaced with a 2′-F, 2′-N or 2′-H substituent. We then tested if this shift in the inflection point of the curve is a specific feature associated with the 2′-ribose modifications at nt −1 rather than a general effect, for example, related to a reduction in the rate of the chemical step (k_chem). We, therefore, analyzed the Sr²⁺ dependence of cleavage rate for the all-ribose substrate at two lower pH values to reduce the rate of k_chem. The inflection point of the curve indeed increased with decreasing pH (Figure 6B), which also holds for cleavage of the 2′-F-ptRNAmeasured at the same three pH values (Figure 6C). These findings suggested that it may be difficult to extract metal ion-specific information based on changes of the inflection point between the stimulatory and the inhibitory phases.

Figure 6.

Processing by M1 RNA of the all-ribose ptRNA^Gly (2′-OH) and variants thereof with a single 2′-fluoro (2′-F), 2′-deoxy (2′-H) or 2′-amino (2′-N) modification at nt −1. (A) Processing rates in the presence of 20 mM Zn²⁺ and varying concentrations of Sr²⁺ at pH 6.65. Inflection points between stimulatory and inhibitory phases of curves are marked by dashed lines. The curve for the all-ribose ptRNA^Gly is identical to that shown in Figure 2C. The inset shows the curves for the 2′-H- and 2′-N-modified substrates at higher resolution. (B) Processing rates for all-ribose ptRNA^Gly at three different pH values. (C) As in (B), but using the 2′-F-modified ptRNA^Gly. For further details, see Materials and Methods.

Our affinity measurements (Figure 3A and Table 1) showed that an enzyme concentration of 5 μM was subsaturating at 20 mM Zn²⁺ and ≪5 mM Sr²⁺. Since we attributed the stimulatory effect of Sr²⁺ at low concentrations to its stabilization of E·S complexes, we suspected that changes in [E] may also affect the inflection point between the stimulatory and the inhibitory phases. Indeed, the Sr²⁺-dependence of processing rate displayed changes in the inflection point between the two phases for the all-ribose as well as the 2′-F-ptRNA when monitored at enzyme concentrations of 5 versus 1.4 μM (Figure 7A and B). We then replotted the data of Figure 7A and B as v₀/v_i (v₀ and v_i correspond to k_obs in the absence and the presence of Sr²⁺, respectively; Figure 7C and D). The best fit of the data was obtained utilizing Equation 8 (see Materials and Methods) based on a model outlined in Figure 8A, which involves two Sr²⁺ ions (or two classes of Sr²⁺ ions); both ions improve substrate affinity in a cooperative manner, but one of the two inhibits substrate conversion in a non-competitive mode with respect to the substrate. The two Sr²⁺ ions may well be those suggested by the Hill coefficient of the binding data in Figure 3B. The fact that both Sr²⁺ ions contribute to the formation of high-affinity E·S complexes is accounted for by introducing the interaction factors α and β in the scheme of Figure 8A. Other models, for example, assuming the involvement of two ‘activating’ and one inhibitory Sr²⁺ ion or predicting that E·S·I and E·S·I(a)·I complexes retain residual reactivity, failed to give satisfactory curve fits of the data.

Mode of inhibition by Sr²⁺

We finally investigated the inhibition mode of Sr²⁺ with respect to the Zn²⁺ by varying the Sr²⁺ concentration between 10 and 40 mM at four different Zn²⁺ concentrations. Under these conditions, the enzyme concentration (5 μM) was assumed to be saturating at all variations of Sr²⁺ and Zn²⁺, even at the combination of 10 mM Sr²⁺ and 20 mM Zn²⁺, where a K_d of ∼250 nM was determined (Figure 3A). Thus, cleavage chemistry was expected to limit the rate of substrate turnover. The Dixon plot of the data (Figure 8B) gave straight lines that intersect on the [I] axis, which is a specific feature of non-competitive inhibition. The point of intersection yields a K_I value of ∼5 mM. The result argues against a direct displacement of catalytic Zn²⁺ by a Sr²⁺ ion at the aforementioned metal ion site [B] (Figure 5).

DISCUSSION

Catalysis in the presence of Zn²⁺

M1 RNA-catalyzed processing in the presence of Zn²⁺ as the only divalent metal ion present in the cleavage assay (termed Zn²⁺ alone conditions in the following) is in contrast to the previous studies (11,15). This discrepancy can be explained by the fact that our study was performed under conditions of E≫S and in the presence of high concentrations of NH₄OAc. With potassium or sodium instead of ammonium salts, we were unable to detect M1 RNA-catalyzed cleavage under Zn²⁺-alone conditions (Figure 2A). One possibility is that Zn²⁺ ions partly replace water ligands with ammonia (26) as a requirement to be able to sustain catalysis by M1 RNA. Although proficient in catalysis, Zn²⁺ is unable to support thermodynamically stable E·S complex formation (Table 1 and Figure 3A). Yet selection of the canonical cleavage site (between nt −1 and +1) was not changed in the presence of Zn²⁺ alone (relative to Mg²⁺ alone; data not shown), despite the very low substrate affinity under Zn²⁺ alone conditions. This shows that low affinity substrate ground state binding not necessarily favors aberrant cleavage (between nt −2 and −1) relative to cleavage at the canonical site. One interpretation is that the high activation barrier difference for aberrant cleavage (at −2/−1) relative to canonical cleavage (−1/+1) is maintained under these conditions. In conclusion, RNase P RNA catalysis with Zn²⁺ as the metal cofactor represents a ribozyme case where the specific transition state is achieved despite a dramatic destabilization of the substrate ground state binding.

We had speculated that the inability of Zn²⁺ to mediate high-affinity substrate binding might enable us to dissect the metal ions that mediate substrate ground state binding from those specifically involved in transition state stabilization in order to define the subset of catalytic metal ions. This, however, turned out to be difficult because saturating enzyme concentrations could not be reached in the absence of a second non-catalytic metal ion, such as Sr²⁺, but Sr²⁺ in turn [as $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$] inhibited catalysis. This resulted in complex velocity versus [Sr²⁺] curves with ascending and descending sections (Figure 2C, 6, 7A and B). Secondary plots of these data gave reasonable fits to a model involving two Sr²⁺ ions (or two classes of Sr²⁺ ions). Both Sr²⁺ ions support substrate binding in a cooperative manner, but one of the two inhibits substrate conversion. In a non-competitive mode with respect to the substrate. As a result, Sr²⁺ stimulated processing at low concentrations by shifting the E·S equilibrium toward complex formation, whereas the inhibitory effect dominated at higher concentrations. Curves of k_obs versus [Sr²⁺] turned out to be highly sensitive to changes in [E] or k_chem (Figures 6 and 7). By analyzing Sr²⁺ inhibition of M1 RNA cleavage with Zn²⁺ as catalytic cofactor in the context of different substrates with 2′-ribose modifications, we had hoped to extract specific information regarding the catalytic metal ion binding to site [B] (Figure 5). Indeed, the βK_I values for Sr²⁺ inhibition derived from the secondary plots in Figure 7C and D are ∼15-fold higher for the 2′-F versus all-ribose ptRNA substrate, while αK_I(a) values are equal within a factor of 2. This finding is consistent with the binding of an Sr²⁺ ion at or near metal ion site [B] with the help of the 2′-OH at nt −1. However, inhibition kinetics (Figure 8B) with the all-ribose substrate favored a model of non-competitive inhibition of Sr²⁺ with respect to Zn²⁺, thus arguing against a mechanism in which the inhibitory Sr²⁺ ion directly displaces a catalytic Zn²⁺ at metal ion site [B]. It, therefore, remains to be defined how Sr²⁺ inhibits the catalytic process with Zn²⁺ as catalytic cofactor.

Our kinetic analyses (Figure 4) have revealed an involvement of two or more Zn²⁺ ions, or classes of Zn²⁺ binding sites, in M1 RNA catalysis. This result is similar to those obtained with Mg²⁺ as the metal ion cofactor (6). These authors concluded that at least three Mg²⁺ ions take part in the catalytic step.

Zn²⁺ has also been explored as a catalytic cofactor in the reaction catalyzed by the B.subtilis holoenzyme (9) with an RNA subunit of the structural type B. Single turnover activity (with 1 μM holoenzyme and 100 mM KCl, pH 8.0) was very low in the presence of 10 mM Zn²⁺ (2.4 × 10⁻³ min⁻¹). However, the addition of 2 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ in combination with only 0.2 mM Zn²⁺ resulted in a processing rate of ∼11 min⁻¹, which was only ∼5-fold lower than the cleavage rate under saturating Mg²⁺ conditions, with Zn²⁺ activating cleavage at lower concentrations than Mg²⁺ (9). The latter is attributable to the fact that Zn²⁺ (as Mn²⁺) is a better Lewis acid than Mg²⁺ [pK_a for the formation of Me[H₂O]₅[OH]⁺ of ∼9.0 versus 11.4 for Mg²⁺; (27)]. These results combined with ours obtained for the E.coli system document that Zn²⁺ is a proficient cofactor of bacterial RNase P (RNA) catalysis.

Comparison with results from the previous studies of M1 RNA

The global conformation of M1 RNA has previously been probed by lead ion-induced hydrolysis in the absence of substrate (17). Cd²⁺ substantially altered the lead hydrolysis pattern of M1 RNA relative to Mg²⁺, while changes were more moderate in the presence of Zn²⁺, which suggested substantial changes of M1 conformation induced by Cd²⁺ but to a lower extent by Zn²⁺ (17). These findings are entirely different from the relative effects of Zn²⁺ and Cd²⁺ on the substrate affinity observed in the work presented here, indicating that Zn²⁺ is much more detrimental to E·S formation than Cd²⁺ at the same concentrations (Table 1). Yet, despite the better performance of Cd²⁺ in E·S formation, Zn²⁺ supports cleavage of the all-ribose ptRNA at a 16-fold higher rate than Cd²⁺ (data not shown) under our standard conditions (Figure 2A, in the presence of 1 M NH₄OAc and 20 mM Me²⁺). This may be related to the fact that the formation of an Me[H₂O]₅[OH]⁺ species required for RNase P catalysis (see below) is favored with Zn²⁺ over Cd²⁺ [pK_a of ∼9.0 for Zn²⁺ versus >10 for Cd²⁺; (27)].

The lead-induced hydrolysis patterns of M1 RNA also suggested that the M1 RNA conformation is rather similar in the presence of Mg²⁺, Mn²⁺, Ca²⁺, Sr²⁺, Ba²⁺ and $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ (17). However, we found that $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ at higher concentrations substantially reduced the fraction of ptRNA substrates capable of binding to saturating concentrations of M1 RNA, which was also observed to some extent for Zn²⁺ (Table 1 and Figure 3A). Likewise, Zn²⁺ and $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ are expected to affect the proportion of catalytically competent M1 RNA, which will be of particular importance when cleavage assays are performed in the presence of limited amounts of ribozyme (E ≪ S). In conclusion, future studies will have to incorporate the differential effects that metal ions (or metal ion mimics) other than Mg²⁺ have on structural equilibria of ribozyme and substrate RNAs as well as E·S complex formation in addition to the catalytic performance.

Failure of Sr²⁺ to support catalysis

Little is known on the binding of Sr²⁺ ions to RNA, but a coordination geometry different from the canonical octahedral Mg²⁺ geometry may be the cause for the failure of Sr²⁺ to activate catalysis by RNase P RNA under standard conditions and its inhibitory mode in the reaction with Zn²⁺ as the metal cofactor. Indeed, a coordination geometry resembling a slightly distorted trigonal prism and involving nine oxygen atoms (four ribose hydroxyl groups and five waters) were observed for a Sr²⁺ ion in the crystal structure of the tRNA^Ala acceptor stem (28). Taking into account that four hydroxyl groups were inner-sphere ligands of this Sr²⁺ ion, whereas inner-sphere coordination of 2′-OH ligands to Mg²⁺ seems to be rare (29), it is an intriguing possibility that Sr²⁺ fails to support M1 RNA catalysis owing to inner-sphere coordination to the 2′-OH at nt −1 of the substrate. A role for this substituent in Sr²⁺ binding is indeed indicated by a weaker inhibitory effect of Sr²⁺ in the context of the ptRNA substrate with a 2′-F modification at nt −1 (Figure 7, see Discussion).

Further information on binding of Sr²⁺ to RNA stems from high-resolution structures of the leadzyme in the presence of Mg²⁺ versus Mg²⁺ plus Sr²⁺ (30). Three Mg²⁺ and three Sr²⁺ ions were identified, the Sr²⁺ ions occupying different sites on the RNA than the Mg²⁺ ions. All three Mg²⁺ ions contacted the RNA duplex via their canonical octahedral hexa-hydration sphere, while ligand spheres of the three Sr²⁺ ions varied in number and did not uniformly consist of inner-shell water molecules. One Sr²⁺ ion ([Sr]3,(30)) had three water molecules, three inner-sphere base or phosphate oxygen ligands, and was 3.8 Å from the oxygen of the 2′-OH at C23 that serves as the nucleophile in the leadzyme reaction after proton abstraction by catalytic Pb²⁺. Sr²⁺ coordination next to the 2′-OH of C23 offered an explanation why Sr²⁺ inhibits catalysis by Pb²⁺ (30). The same Sr²⁺ ion also caused modest but significant local changes in the immediate vicinity of the cleavage site, thereby favoring a ‘pre-catalytic’ over the ‘ground-state’ conformation of the leadzyme. Such local, Sr²⁺-induced changes in the active site of RNase P RNA–substrate complexes may well have contributed to the inhibition effects seen in the RNase P system.

Effects of Sr²⁺ and Zn²⁺ on substrate binding and structure

K_d measurements (Figure 3A and Table 1) were performed with trace amounts of ³²P-labeled ptRNA and varying excess amounts of enzyme using a gel filtration spin column assay (8,23). Zn²⁺ increased the proportion of binding-deficient ptRNA molecules at saturating enzyme concentration (i.e. at the endpoint), a feature that is attributable to Zn²⁺ ions bound to ptRNA. Increasing Sr²⁺ concentrations at constant 20 mM Zn²⁺ largely reduced this binding-deficient ptRNA fraction, suggesting that Sr²⁺ can displace many of the deleterious Zn²⁺ ions from the substrate. The presence of 20 mM Zn²⁺ also caused a constant 2–3-fold increase in K_d over the entire range of tested Sr²⁺ concentrations (5–80 mM, Figure 3A). This indicates that Sr²⁺ is unable to displace Zn²⁺ (or to compensate its deleterious effects) at some sites where Zn²⁺ directly or indirectly impairs high-affinity substrate binding. Since K_d reflects structural properties of enzyme and substrate, the Zn²⁺ binding sites responsible for this K_d increase may be on the substrate and/or enzyme. To understand the structural effects of Zn²⁺ observed in the present study, it is instructive to inspect the Zn²⁺ binding sites detected in yeast tRNA^Phe crystals (31). Five bound Zn²⁺ ions were identified, two of which, Zn(1) and Zn(2), replaced tightly bound Mg²⁺ ions in the U8–U12 region and in the D loop [corresponding to the Mg²⁺ binding sites 1 and 3 in Jovine et al. (32)], one [Zn(3)] overlapping with the weak Mg²⁺ binding site 7 in (32), and the remaining two [Zn(4,5)] being Zn²⁺-specific or transition metal ion-specific sites in base-paired regions. All five Zn²⁺ ions were coordinated tetrahedrally, and four of them were bound by direct coordination to a guanine N7 at positions where the G residue is flanked by a purine residue on its 5′ side. Zn(1) is shifted ∼2 Å relative to Mg²⁺ at this site. Based on these observations, Zn²⁺ may well cause specific changes of tRNA conformation or may occupy novel Zn²⁺-specific sites that disturb ptRNA interaction with M1 RNA. The preference of Zn²⁺ for purine–guanine dinucleotides also in paired regions implies that (i) some Zn²⁺ binding sites may directly perturb E·S contacts involving acceptor and T stems regions and (ii) that effects of Zn²⁺ will be to some extent sequence-specific and thus specific for every individual RNA under investigation.

Effects of $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ on M1 RNA-catalyzed cleavage

The addition of 5 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ to 20 mM Zn²⁺ stimulated ptRNA turnover ∼2-fold, but higher $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ concentrations were inhibitory as observed for Sr²⁺ (Figure 2C and D). Recently, inhibition of the B.subtilis RNase P holoenzyme by $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ with Mg²⁺ as the catalytic cofactor (9) was discussed to indicate that $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ displaces a metal ion for which the ionization or the displacement of a water molecule from the metal hydration shell is required. Here, it is instructive to compare the properties of Sr²⁺ and $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$, which showed similar inhibition effects (Figure 2C and D). Since water ligands can dissociate from the hydration shell of Sr²⁺ (28) but ammine ligands do not dissociate from the inert octahedral complex cation [$\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$] (33), the remaining common feature of the two is the low degree of ionization of water ligands at pH ≤ 7 [pK_a of Sr²⁺ aqua ion = 13.2; (27)] in the case of Sr²⁺ and the complete absence of ionizable water ligands in the case of $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$. This would be consistent with the involvement of a Me[H₂O]₅[OH]⁺ species in the catalytic process (9,15,24).

Effects of $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ on substrate structure and binding

Soaking of yeast tRNA^Gly crystals with Co(NH₃)₆Cl₃ identified three [Co(NH₃)₆]³⁺ complexes which, however, did not replace strongly bound Mg²⁺ ions (34). Two bound to double-helical guanylguanosine sequences (G3/G4 [Co(2)] and G42/G43 [Co(1)]) and the third [Co(3)] to the purine–purine sequence A44/G45, in all cases in the major groove via hydrogen bonding of cis-ammine ligands to the N7 and O6 functions of adjacent purine bases. Additional hydrogen bonding occurred to O4 of U residues and to phosphate oxygens, but no direct metal–nucleotide bonds were observed (34). Interestingly, the binding site for Zn(5) [see above; (31)] overlapped with the site for Co(1), both contacting the N7 and O6 functions of G42 and G43. However, coordination of the tetrahedral Zn(5) involved an innersphere contact to the N7 of G43, and the octahedral Co(1) formed two additional contacts to non-bridging phosphate oxygens at positions 24 and 42 (34).

We observed that only about one-fourth of the ptRNA molecules were capable of binding to M1 RNA at saturating enzyme concentrations in the presence of 80 mM $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ compared with the conditions of 80 mM Sr²⁺ (Table 1). One explanation may be related to the preference of $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ for GG dinucleotides in paired regions (see above) and the multiple presence of such potential binding sites in our ptRNA^Gly (Figure 1). Binding of $\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$ to some sites in the acceptor stem and T arm may prevent crucial contacts to M1 RNA either directly or may perturb the tRNA tertiary fold.