The naturally trans-acting ribozyme RNase P RNA has leadzyme propertie
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《核酸研究医学期刊》
Department of Cell and Molecular Biology, Uppsala University Box 596, Biomedical Centre, SE-751 24 Uppsala, Sweden 1Department of Molecular Biology, Swedish Agricultural University Box 590, Biomedical Centre, SE-751 23 Uppsala, Sweden
*To whom correspondence should be addressed. Tel: +46 18 471 4068; Fax: +46 18 53 03 96; Email: Leif.Kirsebom@icm.uu.se
ABSTRACT
Divalent metal ions promote hydrolysis of RNA backbones generating 5'OH and 2';3'P as cleavage products. In these reactions, the neighboring 2'OH act as the nucleophile. RNA catalyzed reactions also require divalent metal ions and a number of different metal ions function in RNA mediated cleavage of RNA. In one case, the LZV leadzyme, it was shown that this catalytic RNA requires lead for catalysis. So far, none of the naturally isolated ribozymes have been demonstrated to use lead to activate the nucleophile. Here we provide evidence that RNase P RNA, a naturally trans-acting ribozyme, has leadzyme properties. But, in contrast to LZV RNA, RNase P RNA mediated cleavage promoted by Pb2+ results in 5' phosphate and 3'OH as cleavage products. Based on our findings, we infer that Pb2+ activates H2O to act as the nucleophile and we identified residues both in the substrate and RNase P RNA that most likely influenced the positioning of Pb2+ at the cleavage site. Our data suggest that Pb2+ can promote cleavage of RNA by activating either an inner sphere H2O or a neighboring 2'OH to act as nucleophile.
INTRODUCTION
The interaction of divalent metal ions e.g. Mg2+ with the negatively charged backbone of RNA promotes proper folding and therefore plays an important role for RNA function/activity. Ribozymes or catalytic RNAs catalyze a large number of reactions including cleavage of other RNA molecules. In addition to promoting correct folding and facilitating the interaction with the RNA substrate, divalent metal ions are directly involved in the chemistry of RNA mediated cleavage of RNA. But note that certain RNAs e.g. the hammerhead and the hairpin ribozymes function in the absence of divalent metal ions (1).
Binding of metal ions often results in hydrolysis of the RNA backbone, e.g. lead(II)-induced cleavage of RNA (2,3). Cleavage products in metal(II) ion-induced hydrolysis of RNA have 5'OH and 2';3' cyclic phosphate at their ends (Figure 1D). This is also the case when RNA is cleaved by small ribozymes e.g. the hammerhead RNA. For both hammerhead and metal(II) ion-induced cleavage it has been suggested that the 2'OH at the site of cleavage is the active nucleophile (2,4). For the naturally occurring trans-acting ribozyme RNase P RNA, and for other large ribozymes (originating from Group I and Group II introns) that generate 5'P and 3'OH as cleavage products, we have argued that the strategy must be to prevent the 2'OH at the cleavage site from acting as a nucleophile and instead facilitate nucleophilic attack from the other side of the phosphorous center to ensure correct cleavage products (5). Thus, positioning of metal ions in relation to the cleavage site is of fundamental importance to ensure correct cleavage and to suppress unwanted hydrolysis of the RNA.
Figure 1 (A) Structure of the different pATSer variants used. Y indicates U or C at this position, I = inosine, 2AP = 2-aminopurine, DAP = 2, 6-diaminopurine, Pu = purine, dG = deoxyG, ddG = c7deoxyG, Rib = ribavirin, 3 mU = 3-methyl U, denotes that the base at this position was deleted while cs and red arrow = canonical RNase P cleavage site between positions –1 and +1. Blue arrows indicate Pb2+-induced cleavage sites (note that Pb2+ also promoted cleavage at position +2, not indicated in the figure but see text) and in the case of the U–1 variants the blue arrows indicate that cleavage was observed at +1 in addition to cleavage at the other positions. The elips denote the residues that had been deleted in these variants. (B) The structures of guanosine (G; top) and uridine (U; bottom), the red circles indicate the chemical groups that were substituted while the grey area corresponds to the part of G that is missing in ribavirin, for details see text. (C) Model of the RCCA–RNase P RNA interaction (interacting residues underlined). The letters A–C refer to divalent metal ions that have been identified in the substrate and in the P15 loop (for references see text). The A248/N–1 interaction is indicated (D) Suggested mechanism for Me2+-induced e.g. Pb2+ or Mg2+ cleavage of RNA. The Me2+ acts as a general base and activates the 2'OH resulting in 5'OH and 2';3'P as cleavage products (left). In RNase P RNA mediated cleavage available data suggest that Me2+-OH acts as the nucleophile resulting in 5'P and 3'OH as cleavage products (right). Me encircled in red denotes the divalent metal ion.
RNase P RNA is the catalytic subunit of the endoribonuclease RNase P that is responsible for generating the mature 5' termini of tRNA (6,7). Cleavage requires the presence of divalent metal ions, with Mg2+ as the preferred divalent metal ion. However, the presence of other divalent metal ions such as Mn2+ and Ca2+ can also promote cleavage . Also, combinations of divalent metal ions that do not promote cleavage (or do so poorly) when present alone resulted in increased cleavage activity when present in combination, e.g. mixing Sr2+ with Mn2+ or Zn2+ (9,10). This suggests that there is metal ion cooperativity in RNase P RNA mediated cleavage (9). One of the metal ions involved in this cooperativity was suggested to be positioned in the vicinity of the interaction between the 3' end of the substrate and RNase P RNA, the RCCA–RNase P RNA interaction . According to our model, the other metal ion(s) would be positioned at or in the vicinity of the cleavage site and be involved in generating the nucleophile (Figure 1C). Recently, we presented functional and structural evidence that substitution of the base pair at the cleavage site (the +1/+72 bp) influenced Mg2+ binding in its vicinity (12). This finding is in keeping with previous data suggesting that a metal ion is bound in the vicinity of the cleavage site. Earlier data also suggest that a metal ion coordinated H2O acts as the nucleophile (5,13–24). In the present investigation, we were initially interested to study the structural requirements for metal ion binding at the RNase P cleavage site. Therefore, we studied Pb2+-induced cleavage of model RNA hairpin RNase P substrates carrying changes at the cleavage site. Moreover, based on our previous data demonstrating that Pb2+ promotes hydrolysis of model RNA hairpin substrates near the RNase P cleavage site in the absence of RNase P RNA (21) and that combinations of metal(II) ions that do not promote cleavage (or do so poorly) when present alone can result in increased cleavage activity, we were also interested to investigate whether Pb2+ in combination with other divalent metal ions can promote RNase P RNA mediated cleavage.
Here we present data suggesting that the presence of a purine at the +1 position in the substrate suppresses Pb2+-induced cleavage at this position. However, a guanosine at +1 resulted in M1 RNA (RNase P RNA derived from Escherichia coli) mediated cleavage at the correct position in the presence of Pb2+ and Sr2+ or as the only divalent metal ions. Thus, RNase P RNA has leadzyme properties. The observation that Sr2+ or did not promote cleavage alone is in keeping with a model that Pb2+ is responsible for activating the nucleophile and we identified residues in both RNase P RNA and in the substrate that influence cleavage in the presence of Pb2+. Our findings provide experimental evidence for the model suggesting that the establishment of the RCCA–RNase P RNA interaction results in a re-coordination of Mg2+ positioned in the vicinity of the cleavage site (25).
MATERIALS AND METHODS
Preparation of substrates and RNase P RNA
The various pATSer derivatives were purchased from Dharmacon, USA, purified, labeled at the 5' end and gel purified according to standard procedures as described elsewhere (11,26,27). The RNase P RNA variants were generated as run-off transcripts using T7 DNA-dependent RNA polymerase (27,28).
Assay conditions and determination of the kinetic constants under single turnover conditions
The assays were performed under single turnover conditions at pH 6.1 or 7.2 (as indicated) in Buffer B: 50 mM Bis-Tris Propane, 5% (w/v) PEG 6000, 100 mM NH4Cl and at different concentrations as indicated. All reactions were performed at 37°C. Reaction products were separated on denaturing 20–22% (w/v) polyacrylamide gels and quantified using Phosphorimager (Molecular Dynamics 400S) as described elsewhere (5). The final concentrations of RNase P RNA and substrate were <20 μM and 3.2 μM, respectively.
The kinetic constants kobs and kobs/Ksto (= kcat/Km) were determined under saturating single turnover conditions at pH 6.1 in Buffer B as described previously (5,12) at: = 40 mM; = 40 mM and = 8 mM; = 40 mM and = 8 mM; = 160 mM and = 8 mM. The final concentration of substrate was <20 nM whereas the concentration of RNase P RNA was varied between 0.025 and 6.4 μM dependent on substrate and RNase P RNA variant. For the calculations we used the 5' cleavage fragments and the time of cleavage was adjusted to ensure that we were in the linear part of the curve of kinetics. The kinetic constants were obtained by linear regression from Eadie–Hofstee plots.
Binding assay conditions
Spin columns were used to determine the apparent equilibrium dissociation constant (appKd) as described elsewhere (5,12) in Buffer C: 50 mM MES at pH 6.0, 0.8 M NH4OAc, 0.05 %(w/v) Nonidet P40, 0.1% (w/v) SDS and different concentration of CaCl2 or SrCl2 as indicated. The final concentration of substrate was <20 nM and was varied between 0.025 to 6.4 μM (concentration range depended on RNase P RNA variant). The appKd was determined by using OriginPro 7.0 software and the equation fc = ft x free/(Kd + free), where fc represent fraction of substrate in complex with RNase P RNA and ft = maximum fraction of substrate able to bind RNase P RNA, i.e. end point.
Lead(II)-induced cleavage conditions
The different 5' end labeled pATSer derivatives were subjected to Pb2+-induced cleavage in Buffer A: 50 mM Tris–HCl pH 7.2, 5% (w/v) PEG 6000, 100 mM NH4Cl, 40 mM MgCl2 (or SrCl2) and PbOAc at a final concentration of 4 mM. The substrates were preincubated in Buffer A in the absence of PbOAc for 10 min at 37°C, incubated for 5 min after addition of PbOAc and the cleavage products were analyzed by gel-electrophoresis as described elsewhere (21,27).
Determination of the site of cleavage
Cleavage at +1 was inferred by comparing the mobility of the 5' cleavage fragments generated in the presence of Pb2+/Sr2+ and Mg2+ (see above). To verify the presence of 5' phosphate at the 5' termini of the cleaved product (internally labeled with GTP), the large cleavage product was gel purified and subjected to digestion with RNase T1, RNase T2 and pancreatic RNase A as described previously (29). Thin-layer chromatography according to Nishimura (30) was used to detect the 5' phosphate-labeled nucleotide i.e. pGp.
RESULTS
A purine at +1 suppresses metal induced cleavage at this position
The pATSerCG substrate is a well-characterized RNase P substrate that is cleaved at several positions in the 5' leader in the presence of Pb2+ and absence of RNase P RNA (21). A similar type of substrate was also used elsewhere and it was demonstrated that Mg2+ induced cleavage in the 5' leader the vicinity of the RNase P cleavage site (15). Hence, to investigate the structural requirements for positioning of a metal(II) ion at the RNase P cleavage site, we introduced changes at positions –1, +1 and/or +72 in pATSer (Figure 1A; the residue that pairs with residue +1 corresponds to residue +72 in tRNA and will therefore be referred to residue +72). These pATSer variants were subjected to Pb2+-induced cleavage. As shown in Figure 2, introduction of a U or C at +1 resulted in significant cleavage at +1 irrespective of identity of the residue at +72 or –1. By contrast, a significant reduction in Pb2+-induced cleavage at +1 was observed due to the presence of purine derivatives at this position. Moreover, our data suggested that base pairing between U+1 and the base at +72 did not influence Pb2+-induced cleavage at +1 (Figure 2, compare cleavage of U+1/A+72, U+1/DAP+72, U+1/C+72 and 3mU+1/A+72 variants). Neither did substitution of N7 of G+1 with c7 as was evident when Pb2+ cleavage of pATSerUGG/C, pATSerUGdG/C and pATSerUG(c7dG)/C was compared (data not shown; underlined residues corresponds to residues G+1 and C+72) or in the absence of the 3' terminal sequence G+73CCAC (Figure 1A). A significant Mg2+-induced cleavage at +1 was also observed due to replacement of G+1/C+72 with U+1/A+72 (data not shown). Surprisingly, introduction of ribavirin at +1 resulted in a similar suppression of Pb2+-induced cleavage at +1 compared to when the substrate carried a purine at this position.
Figure 2 Histograms showing Pb2+-induced cleavage of the different substrates in the absence of RNase P RNA as indicated. The distribution of the frequency of cleavage at –1, +1 and +2 were calculated and the relative distribution in percent was plotted as indicated. gccac indicate residues that were deleted in these pATSerUG derivatives as illustrated in Figure 1.
The 5' cleavage products generated as a result of addition of Pb2+ migrated slightly slower compared to the migration of the 5' cleavage fragments produced in the M1 RNA mediated cleavage reaction (see below Figure 3A and data not shown). In addition, the Pb2+ induced 5' cleavage products could not be labeled at their 3' ends with pCp (data not shown). These findings are expected given that metal(II)-induced cleavage generates cleavage product ends with 5'OH and 2';3' cyclic phosphates (2,3). Analysis of the 5' end of the larger cleavage product by thin-layer chromatography (i.e. the presence of nucleotides with a 5' phophate could not be detected; Figure 3B upper panel) is in agreement with these findings.
Figure 3 (A) M1 RNA mediated cleavage of pATSerUGG/C, pATSerUGIno/C and pATSerUG2AP/U under single turnover conditions at pH 7.2 in the presence of 8 mM Pb2+ and 40 mM Sr2+ as indicated. Two different M1 RNA variants (3.2 μM final concentration; final concentration of substrate 0.04 μM), wild-type M1 RNA (the number 3 lanes) and M1C294 RNA (the number 4 lanes) were used. Two controls, cleavage of the substrates in the absence of M1 RNA and ± tRNA were included (no tRNA added the number 1 lanes while in the number 2 lanes tRNA was added). The filled circles indicate M1 RNA mediated cleavage at –1. The cleavage reaction was terminated after 5 min (for details see text). The different fragments as a result of cleavage at –2, –1 and +1 are shown. (B) Two-dimensional thin-layer chromatography according to Nishimura (30) demonstrating the presence of 5' pGp (in red) as a result of M1 RNA mediated cleavage in the presence of Pb2+. Internally labeled GTP pATSerUGG/C was incubated in the presence of Pb2+/Sr2+ and in the absence and in the presence of M1 RNA as indicated. The large cleavage product was isolated and subjected to RNase digestion as outlined in Materials and Methods. For further experimental details see text. (C) and (D) M1 RNA mediated cleavage expressed as rate of cleavage in % per min as a function of increasing concentration of Sr2+ (C) and Pb2+ (D) using wild-type M1 RNA and M1C294 RNA as indicated. When Sr2+ was increased the Pb2+ concentration was kept constant at 8 mM likewise when we varied the Pb2+ concentration the Sr2+ concentration was kept constant at 40 mM. The experiments were performed at pH 6.1 at 37°C and the concentrations of M1 RNA and substrate were 3.2 μM and 40 nM, respectively. (E) M1 RNA mediated cleavage expressed as relative cleavage at +1 in % as a function of increasing concentration of as indicated using wild-type M1 RNA. The Pb2+ concentration was 8 mM. The experiment was performed in Buffer B at pH 7.2 at 37°C and the concentrations of M1 RNA and substrate were 3.2 μM and 40 nM, respectively.
We conclude that the local structural architecture around the +1 residue influence the positioning of Pb2+ (or Mg2+) in its vicinity. More specifically we suggest that the presence of a purine at +1 prevents substantial metal induced cleavage at +1 and that the Hoogsteen surface of purine plays an important role. The observed cleavage at +1 for the U+1 and C+1 derivatives could be due to the electronegative environment generated by the oxygen at position 2 of U or C in the shallow groove (see also below). In all other cases tested there is either an exocyclic amine or no chemical group in the shallow groove at this position in the substrate.
RNase P RNA has leadzyme properties
The presence of Pb2+ induces hydrolysis of the phosphate backbone at the RNase P cleavage site (and in its vicinity) in the precursor substrate while combinations of metal ions that do not promote cleavage by themselves (or do so poorly) results in increased RNase P RNA mediated cleavage (9). Pb2+ was not included in our previous studies since we argued that addition of Pb2+ results in cleavage of RNase P RNA as well as other RNAs in particular at pH > 7 (2,3,9). Nonetheless, we decided to investigate whether RNase P RNA can mediate cleavage at the correct position in the presence of Pb2+. Conceivably this would give us a tool to identify factors important for generating 5'P and 3'OH as cleavage products on the one hand, and 5'OH and 2';3' cyclic phosphate on the other, where the former cleavage products are hallmarks for RNase P RNA promoted cleavage and the latter the signature for Me2+- (here Pb2+-) induced cleavage . Addition of Pb2+ will result in cleavage of both M1 RNA and the substrate pATSer at specific as well as unspecific positions (3,7). Thus, cleavage conditions had to be short to minimize RNA degradation by Pb2+ but long enough to be able to detect RNase P RNA mediated cleavage. To achieve this objective we used E.coli RNase P RNA, M1 RNA and pATSerUGG/C as substrate. The pATSerUGG/C substrate (referred to as ‘wild type’) has in our previous studies been demonstrated to be as efficiently cleaved as precursor tRNAs (5,11,12). We preincubated M1 RNA for 10 min at 37°C in the presence of various concentrations of Sr2+ to allow M1 RNA to fold into an active conformation. No difference in the conformation of M1 RNA due to replacement of Mg2+ with Sr2+ was observed using Pb2+-induced cleavage to monitor conformational changes (21). Note also that Sr2+ alone does not promote M1 RNA mediated cleavage under these conditions . The substrate was added (amount added to ensure single turnover conditions) and after an additional incubation for 5 min Pb2+ was added to a final concentration of 8 mM (or as indicated). The cleavage reaction was terminated after 5 min and the cleavage products were separated by gel-electrophoresis (see Materials and Methods). Surprisingly, we observed M1 RNA mediated cleavage at the correct position +1 under these conditions with an optimum cleavage rate at 8 mM Pb2+ and 40 mM Sr2+ (Figure 3A). That cleavage was indeed mediated by M1 RNA was confirmed by employing thin-layer chromatography to demonstrate the presence of pGp at the 5' end of the cleavage product (Figure 3B). We also tried the combination Pb2+ and . Here we had to reduce the concentration of significantly to detect M1 RNA mediated cleavage since higher concentrations of resulted in inhibition (Figure 3E). The lower concentration of is rationalized by that binds to RNA with higher affinity compared to e.g. Sr2+ (21). Consequently, at higher concentration of , most likely displace Pb2+ and thereby cleavage is inhibited (see also below). Taken together, these findings are in keeping with our study where we observed cleavage when we mixed Sr2+ or with Zn2+ (9). In this context we note that Taira and coworkers (4) have studied the hammerhead cleavage reaction in the presence of different metal ions. Their data suggest that La3+ can act both as an activator and inhibitor of cleavage .
A possibility is that the observed activities are due to contamination of Mg2+ or Ca2+. However, in the presence of 40 mM Sr2+ and 0.1 mM Mg2+ we did not observe M1 RNA mediated cleavage (data not shown). Also, cleavage in the presence of 15 mM Ca2+ and 25 mM Sr2+ reduce the efficiency of cleavage >100-fold compared to cleavage in the presence of 15 mM Mg2+ and 25 mM Sr2+ (9). Hence, since addition of 0.1 mM Mg2+ did not result in cleavage we do not expect that addition of Ca2+ would. These data argue against contamination of Mg2+ and Ca2+.
Replacing pATSerUGG/C with a conventional tRNA precursor, pSu3, also resulted in cleavage at the correct position at +1 in the presence of Pb2+ and Sr2+ (data not shown). Finally, Pb2+ induced cleavage of 32P-labeled M1 RNA showed very little degradation of M1 RNA after 5 min in the presence of Pb2+ (data not shown).
We conclude that M1 RNA mediated cleavage can be promoted in the presence of Pb2+ and Sr2+ (or ), where Sr2+ and do not promote cleavage when present alone. Thus, M1 RNA has leadzyme properties. This finding is in keeping with a model that Pb2+ is involved in generating the nucleophile i.e. corresponding to MeA in Figure 1C. Given this, the MeB binding site in the M1 RNA substrate (RS-) complex would most likely be occupied by Sr2+ (or ) since a Pb2+ at this position would result in hydrolysis of the M1 RNA phosphate backbone in its vicinity .
The leadzyme-like activity is not a unique feature of M1 RNA
To investigate whether leadzyme-like activity could be observed for other RNase P RNAs we replaced M1 RNA with RNase P RNA derived from Mycoplasma hyopneumoniae, Hyo P RNA (32) and Yersinia pestis, Yer P RNA (Figure 4; B. M. F. Pettersson and L. A. Kirsebom, unpublished data). The former is a type B RNase P RNA and the latter is closely related to M1 RNA i.e. type A. While Hyo P RNA did not mediate cleavage under these conditions, partly due to poor substrate binding (data not shown), Yer P RNA promoted cleavage of pATSerUGG/C at the correct position +1 in the presence of Pb2+/Sr2+ although less efficiently compared to M1 RNA (Figure 5). Both Hyo P RNA and Yer P RNA cleaved pATSerUGG/C efficiently and correctly in the Mg2+ alone cleavage reaction i.e. 40 mM Mg2+ (Figure 5; data not shown for Hyo P RNA). Moreover, the appKd value for Yer P RNA indicated no apparent difference in substrate binding compared to M1 RNA (see below; Table 1). Thus, the leadzyme-like activity is not a unique feature of M1 RNA.
Figure 4 The predicted secondary structure of Yersinia pestis RNase P RNA according to Brown (48). The positions/region of the different changes that were introduced are highlighted in red.
Figure 5 Cleavage rates under single turnover conditions at pH 6.1 as expressed in percentage of cleavage as a function of time for the different RNase P RNA variants as indicated where Yer P represents RNase P RNA derived from Yersinia pestis. The upper panel shows cleavage in the presence of 8 mM Pb2+ and 40 mM Sr2+ and the lower panel in the presence of 40 mM Mg2+. The concentrations of M1 RNA and substrate were 3.2 μM and 40 nM, respectively.
Table 1 Summary of the kinetic constants kobs and kobs/Ksto, and the apparent binding constants, appKd for various RNase P RNAs and pATSerUGG/C at Me2+ concentrations as indicated
Determinations of the apparent binding constant, appKd and the rate constant kobs
Next we determined the apparent binding constant, appKd, at two different Sr2+ concentrations 40 and 160 mM at pH 6.0. This was followed by determinations of the rate constant kobs under single saturating conditions at pH 6.1. At this pH the rate of cleavage is linearly dependent on in the presence of 8 mM Pb2+ and 40 (or 160 mM) mM Sr2+ suggesting that chemistry is rate-limiting at this pH . The linear dependence on is also consistent with that a hydroxyl ion is acting as the nucleophile . To be able to evaluate Pb2+ as a promoter of RNase P RNA mediated cleavage we also determined kobs in the presence of 8 mM Mg2+ and 40 mM Sr2+ at pH 6.1. The results are shown in Table 1.
Comparing appKd for wild-type M1 RNA determined in the presence of Ca2+ or Sr2+ indicated that pATSerUGG/C binds with 10- and 2.5-fold reduced affinity at 40 or 160 mM Me2+, respectively, in the presence of Sr2+. The rate of cleavage at position +1 in the presence of Pb2+ was only reduced 6-fold compared to cleavage in the presence of Mg2+ under the same conditions indicating that Pb2+ promoted cleavage with reasonable rates. Moreover, increasing the Sr2+ concentration resulted in better binding (20-fold) of the substrate but an almost 10-fold reduction in the rate of cleavage. Based on the model that Pb2+ generates the nucleophile this suggested that Sr2+ presumably had replaced Pb2+ in the vicinity of the cleavage site i.e. MeA (Figure 1).
Structural architecture of the cleavage site affects positioning of Pb2+
The observation that Pb2+ is positioned close to the cleavage site and likely involved in generating the nucleophile gives us a handle to identify residues and chemical groups in M1 RNA as well as in the substrate influencing RNase P RNA mediated cleavage promoted by Pb2+ (and most likely Mg2+). To address which chemical groups at the cleavage site are important for efficient cleavage in the presence of Pb2+, we cleaved pATSer variants with inosine (and C+72) and 2-aminopurine (and U+72) at +1, respectively (Figure 1A). Here we observed that cleavage of the ‘inosine’ variant was significantly reduced while the level of cleavage of the latter was not significantly different compared to cleavage of the ‘wild-type’ substrate, pATSerUGG/C (Figure 3A). This suggested that the exocyclic amine at position 2 in guanosine plays an important role possibly influencing positioning of Pb2+. The importance of the exocylic amine at this position is consistent with previous findings . Note also that we observed M1 RNA mediated cleavage at –1 in particular using M1C294 RNA (see below). We also tried to cleave the substrate where G+1/C+72 had been replaced with U+1/A+72 in the presence of Pb2+/Sr2+ but no cleavage was observed under these conditions most likely due to this substrate being a poor substrate for M1 RNA mainly due to its influence on metal ion binding in its vicinity . These data suggest that the structural environment at the cleavage site and in particular the exocyclic amine positioned in the shallow groove influenced Pb2+/Sr2+ promoted cleavage. We argue that these results are due to a change in the positioning of the Pb2+ involved in activating the nucleophile.
Structural architecture of ‘the +73/294 interaction’ affects positioning of Pb2+
We recently provided experimental evidence that there is cross talk between ‘the +73/294 interaction’ and the cleavage site. During this process metal ions are suggested to play important roles . Hence, we asked whether a change in the structural architecture of the +73/294 interaction influences the rate of cleavage in the Pb2+-promoted reaction. Based on our model that Pb2+ is located close to the cleavage site (see above) we reasoned that this would give information about whether the positioning of this Pb2+ ion(s) is affected or not. Thus, we used a mutant M1 RNA where U294 had been replaced with a C, M1C294 RNA, resulting in a G+73/C294 interaction instead of a G+73/U294 in the RS-complex (Figure 1B). The ground state binding (appKd at 40 mM Ca2+) of this mutant is very similar compared to the wild-type (Table 1). Moreover, as shown in Table 1 in the Mg2+ alone reaction the rate of cleavage of pATSerUGG/C was only modestly reduced in keeping with our previous data (5). By contrast in the presence of Sr2+, appKd was determined to be 6.5- to 2.5-fold lower relative to wild-type at 40 and 160 mM Sr2+, respectively, indicating an improved ground state binding for this mutant in the presence of Sr2+. Cleavage under single turnover conditions as a function of revealed that the C294 mutant cleaved the substrate more efficiently compared to the wild-type. This was observed to be the case irrespective of whether Mg2+ or Pb2+ promoted cleavage was studied (Figure 4; data only shown for the case when 8 mM Pb2+ was used). This was further corroborated when we determined kobs for M1C294 RNA under the conditions indicated in Table 1. Here we observed a 2.6-fold increase at 40 mM Sr2+/8 mM Mg2+ and a 4.1-fold increase at 40 mM Sr2+/8 mM Pb2+. At 160 mM Sr2+/8 mM Pb2+, we observed only a 1.8-fold increase relative to wild-type again indicating (see above) that higher concentration of Sr2+ inhibits the rate of cleavage under these conditions. These findings suggested that changes of the structural architecture of the +73/294 interaction influence metal ion binding most likely in its vicinity (MeA in Figure 1C; see also below). We argue that as a consequence, this affects the rate of cleavage possibly due to a repositioning of the metal ion involved in generating the nucleophile. This further indicates the role of metal ions bound at different sites in RNase P RNA mediated cleavage .
Identification of a residue in RNase P RNA affecting positioning of Pb2+
The efficiency of the Pb2+/Sr2+ promoted cleavage for Yer P RNA was lower compared to that of M1 RNA. The secondary structures of these two RNase P RNAs are very similar and differences were identified in four regions: C125A, U333G, U364C (residue in M1 RNA underlined) and AUAA in M1 RNA (see Figure 4) while Yer P RNA carries GCAU at this site. This gave us a way to identify other residues/regions in RNase P RNA potentially involved in positioning Pb2+ in the vicinity of the cleavage site. Thus, we generated four Yer P RNA variants carrying C125, U333, U364 and AUAA269 (M1 RNA numbering; Figure 4) to investigate whether the Yer P RNA activity could be rescued. As shown in Figure 5, only the C to U replacement at 364 resulted in an increased cleavage activity while the other changes did not. This was observed both in the Pb2+/Sr2+ and Mg2+ promoted reactions. Neither of these changes affected ground state binding (Table 1). These findings raise the possibility that the residue at 364 in Yer P RNA influenced positioning of the Pb2+ in the vicinity of the cleavage site.
DISCUSSION
Lead(II) ion is an efficient inducer of hydrolysis of the phosphate backbone of RNA generating 5'OH and 2';3'P as cleavage products (2,3). Here we showed that RNase P RNA is able to cleave its substrate at the canonical site in the presence of the Pb2+ and Sr2+ or .
The pKa value for Sr2+ is significantly higher compared to Mg2+ and Ca2+ that do promote RNase P RNA mediated cleavage (2,7). In a previous study Sr2+ was demonstrated to promote cleavage at elevated pH, pH 9 (14). Thus, one could argue that Pb2+ binding in the RS-complex ‘activates’ Sr2+ that subsequently results in generation of the nucleophile. Here we observed RNase P RNA mediated cleavage in the presence of Pb2+/Sr2+ at a significantly lower pH (6.1 and 7.2). When present alone, Sr2+ (or ) has not been demonstrated to promote cleavage by RNase P RNA under these conditions, i.e. at pH 7.5 . Also, an increase in the Sr2+ concentration resulted in inhibition of cleavage. Moreover, mixing Pb2+ with resulted in RNase P RNA mediated cleavage and increasing concentration of also inhibited cleavage. Together this argues against ‘activation’ of Sr2+. Rather the role of Sr2+ (or ) is suggested to promote RNA folding and facilitate/stabilize the interaction with the substrate. Note that available data suggest that RNase P RNA is not correctly folded as well as that it is efficiently cleaved in the presence of only Pb2+ (21). Our data also argues against the presence of contamination of e.g. Mg2+ in our solutions. We conclude that our data are consistent with a model where Pb2+ most likely is responsible for generating the nucleophile. To conclusively demonstrate that this is indeed the case will require detailed structural analysis of the RNase P RNA substrate complex.
Compared to Mg2+ that is the preferred metal(II) ion in promotion of RNase P RNA mediated cleavage, Pb2+ shows different physico-chemical properties but their preferred coordination numbers are similar: Pb2+ 6 to 8 and Mg2+ 6 (36). Moreover, although the softer Pb2+ ion is larger than Mg2+, Pb2+ and Mg2+ bind to overlapping sites (2). This, together with that Pb2+ can act as an efficient general base (pKa 7.8) as well as the similarity in coordination numbers might be the only reasons to why Pb2+ can promote RNase P RNA mediated cleavage. Whether other criteria also has to be fulfilled remains to be elucidated. But, unlike lead(II)-induced hydrolysis or that by the in vitro evolved leadzyme which generate 5'OH and 2';3'P as cleavage products (37), Pb2+-promoted cleavage by RNase P RNA resulted in 5'P and 3'OH. Therefore, the mechanism of cleavage has to be different and, in the case of RNase P RNA the nucleophilic attack has to come from the opposite side relative to the positioning of the 2'OH at the cleavage site (Figure 1C). Available data suggest that H2O is the nucleophile in RNase P RNA mediated cleavage (13,17,24). In keeping with this we demonstrated that cleavage by RNase P RNA in the presence of Pb2+/Sr2+ is linearly dependent on (see above). Thus, most likely a H2O ligand is also the nucleophile in RNase P RNA cleavage in the presence of Pb2+ i.e. Pb2+ acts as a general base. To our knowledge this is the first time that Pb2+ has been suggested to activate H2O to act as a nucleophile in RNA mediated cleavage of RNA. To conclude, our present finding that Pb2+ likely act as a catalytic metal ion in M1 RNA mediated cleavage gives us a handle to identify factors/chemical groups that promote M1 RNA mediated cleavage and suppress cleavage of the phosphate backbone in the 5' leader due to activation of neighboring 2'OH groups and thereby increase our understanding of cleavage of RNA by RNA.
Close and distant residues affect positioning of Pb2+ in the RS-complex
Based on the discussion above Pb2+ promotes cleavage at and in the vicinity of the RNase P cleavage site both in the absence and presence of RNase P RNA (Figure 3), where efficient cleavage at the canonical site (with correct cleavage products and G at +1) was only observed in its presence. Hence, it is conceivable that Pb2+ positioned in the vicinity of the cleavage site is re-positioned as a result of formation of the RS-complex arguing for the possibility that the metal ion responsible for generating the nucleophile is associated with the substrate . The importance of the structural architecture of the +1/+72 for metal ion binding in the vicinity of the cleavage site as well as the finding that the presence of the exocyclic amine at position 2 of the guanosine at +1 is important for M1 RNA mediated cleavage in the presence of Pb2+ is in keeping with this model . However, we cannot exclude the possibility that the metal(II) ion(s) involved in generating the nucleophile is recruited from the solution or is associated with RNase P RNA e.g. Me2+ bound in the P4 helix (8).
Irrespective of whether MeB (Figure 1C) is associated with the substrate, RNase P RNA or is recruited from the solution, our data indicate that the structural architecture of the +73/294 interaction influenced the rate of cleavage promoted in the presence of Pb2+ as well as metal(II) binding in its vicinity. This is consistent with our previous findings where we showed that the structural architecture of the +73/294 interaction, that is part of the RCCA–RNase P RNA interaction (25), affects the charge distribution at the cleavage site, ground state binding, rate of cleavage and metal ion binding in its vicinity . Thus, our present findings provide further evidence for cross talk between the +73/294 interaction and the site of cleavage and experimental evidence for the involvement of metal ion(s) in this process.
Our data also indicate that U364 in M1 RNA is likely to influence positioning of Pb2+ in the vicinity of the cleavage site. The significance of this finding is not clear yet but it might indicate that either that this region is positioned in the vicinity of the cleavage site or that this change results in distance effects e.g. by affecting positioning of the metal(II) ions positioned in the P4 helix . However, based on the recent RNase P RNA crystal structures the former alternative is less likely (39,40). Clearly we need to investigate this in further detail but nonetheless this finding opens for new possibilities and approaches using hybrid RNase P RNA molecules in our efforts to understand the complex orchestration in RNase P RNA mediated cleavage. In this context we note that the classical double mutant M1 RNA ts709 carries a G to A change at 365. However, no phenotype has been linked to this change [(41–43); L. A. Kirsebom and S. Altman, unpublished data). Thus, our findings raise the possibility that this mutation indeed influences M1 RNA mediated cleavage by affecting positioning of the metal ion(s) involved in activating the nucleophile.
Structural evidence for positioning a metal(II) ion at the RNase P cleavage site
Our present and previous data clearly indicate that a divalent metal ion(s) is positioned close to the RNase P cleavage site (5,13–24). The high-resolution structure of the RNase P cleavage site is not yet available. However, we noticed that the structures of the ends of the leadzyme are almost identical to the sequences at and in the vicinity of the pATSerCGG/C and pATSerCGU/A cleavage sites with the exception that in the case of the ‘G+1/C+72’ variant the C–1 is single stranded . But in another high-resolution structure of SRP RNA in which the structure at one of the ends is also identical to the sequence of the pATSerCGG/C cleavage site the C–1 is base paired to a G as in pATSerCGG/C . These structural studies suggested that a hexahydrated Mg2+ is bound in the deep groove in the vicinity of the base pairs that would correspond to G+1/C+72 and U+1/A+72, respectively, in our substrates. Superimposing the three Mg2+ binding sites revealed no apparent structural differences. But, the positioning of Mg2+ is suggested to be shifted >7 ? due to the G+1/C+72 to U+1/A+72 replacement, while formation of the ‘C–1/G+73’ base pair only gives a shift of 2.2 ?. In our substrates this would correspond to the Mg2+ near the cleavage site. This supports our functional data suggesting that U/A at +1/+72 affects Pb2+ as well as Mg2+ binding most likely in the vicinity of the cleavage site . Hence, we suggest that there is a functionally important Mg2+, most likely in the hexahydrated state, positioned in the deep groove at the cleavage site. Based on this model it is apparent that hexahydrated Mg2+ is positioned close to the cleavage site (7–8 ?) and to the phosphorous centers of +1, –1 and –2 (Figure 6). Assuming that this is the metal ion that is involved in generating the nucleophile (see above) a repositioning has to occur as a result of the interaction with RNase P RNA to ensure cleavage at +1 i.e. positioning of the nucleophile for an in line attack on the +1 phosphorous center. During this conformational rearrangement, the data suggest that the ‘73/294 interaction’, residue 364 and the exocyclic amine of G+1 in the substrate play important roles (see above). At the same time, attack on the phosphorous center at –1 and –2 should be avoided. In the case of cleavage in the presence of Pb2+ this is not observed due to that Pb2+ is very efficient to activate the neighboring 2'OH to act as a nucleophile while Mg2+ is significantly less efficient (2). This rationalizes why cleavage at other positions within the 5' leader under Pb2+/Sr2+ conditions even in the presence of M1 RNA was observed.
Figure 6 The three dimensional structures derived from the RNA leadzyme (43) and SRP RNA (44) superimposed where the upper representation is viewed from the side and the lower from the top. Two structures, I and III, were taken from the RNA leadzyme (to the left in the figure) whereas structure II was derived from the SRP RNA. The hexahydrated Mg2+ ions are designed B1, B2 and B3 where B1 corresponds to the Mg2+ located in the SRP RNA (II), while B2 and B3 are the Mg2+ ions located in the vicinity of ‘G+1C+72’ (structure I) and ‘U+1A+72’ (III) variants in the RNA leadzyme, respectively. The dashed arrows in parenthesis in the left half of the figure indicate the sites that correspond to the canonical RNase P cleavage site. The red arrows mark the phosphorous atom that corresponds to the one to be attacked in RNase P RNA mediated cleavage. The residues that would correspond to C–1 and G+73 in our pATSer substrate derivatives are indicated in the three dimensional structure representations.
Concluding remarks
RNase P RNA is the first natural ribozyme demonstrated to show leadzyme-like activity. Hence, our findings raise the question whether other ribozymes can mediate catalysis using Pb2+. In this context we note that Mg2+ and Mn2+ promote correct splicing while Mn2+ also induce cleavage at an additional site in the Group I intron. However, Pb2+ does not promote splicing but induce cleavage at the additional site due to that Pb2+ do not promote correct folding (46,47). In view of our present findings it would be of interest to test whether Pb2+ in combination with other divalent metal ions e.g. Sr2+ can promote Group I intron splicing. To the best of our knowledge this has not been tested.
ACKNOWLEDGEMENTS
We thank our colleagues for discussions in particular Drs F. Darfeuille and M. Br?nnvall. Drs S. Dasgupta, D. Hughes and A. Virtanen are acknowledged for critical reading of the manuscript. Finally we thank Ms U. Lustig for technical assistance. This work was supported by a grant to LAK from the Swedish Natural Research Council. Funding to pay the Open Access publication charges for this article was provided by Swedish Natural Research Council.
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*To whom correspondence should be addressed. Tel: +46 18 471 4068; Fax: +46 18 53 03 96; Email: Leif.Kirsebom@icm.uu.se
ABSTRACT
Divalent metal ions promote hydrolysis of RNA backbones generating 5'OH and 2';3'P as cleavage products. In these reactions, the neighboring 2'OH act as the nucleophile. RNA catalyzed reactions also require divalent metal ions and a number of different metal ions function in RNA mediated cleavage of RNA. In one case, the LZV leadzyme, it was shown that this catalytic RNA requires lead for catalysis. So far, none of the naturally isolated ribozymes have been demonstrated to use lead to activate the nucleophile. Here we provide evidence that RNase P RNA, a naturally trans-acting ribozyme, has leadzyme properties. But, in contrast to LZV RNA, RNase P RNA mediated cleavage promoted by Pb2+ results in 5' phosphate and 3'OH as cleavage products. Based on our findings, we infer that Pb2+ activates H2O to act as the nucleophile and we identified residues both in the substrate and RNase P RNA that most likely influenced the positioning of Pb2+ at the cleavage site. Our data suggest that Pb2+ can promote cleavage of RNA by activating either an inner sphere H2O or a neighboring 2'OH to act as nucleophile.
INTRODUCTION
The interaction of divalent metal ions e.g. Mg2+ with the negatively charged backbone of RNA promotes proper folding and therefore plays an important role for RNA function/activity. Ribozymes or catalytic RNAs catalyze a large number of reactions including cleavage of other RNA molecules. In addition to promoting correct folding and facilitating the interaction with the RNA substrate, divalent metal ions are directly involved in the chemistry of RNA mediated cleavage of RNA. But note that certain RNAs e.g. the hammerhead and the hairpin ribozymes function in the absence of divalent metal ions (1).
Binding of metal ions often results in hydrolysis of the RNA backbone, e.g. lead(II)-induced cleavage of RNA (2,3). Cleavage products in metal(II) ion-induced hydrolysis of RNA have 5'OH and 2';3' cyclic phosphate at their ends (Figure 1D). This is also the case when RNA is cleaved by small ribozymes e.g. the hammerhead RNA. For both hammerhead and metal(II) ion-induced cleavage it has been suggested that the 2'OH at the site of cleavage is the active nucleophile (2,4). For the naturally occurring trans-acting ribozyme RNase P RNA, and for other large ribozymes (originating from Group I and Group II introns) that generate 5'P and 3'OH as cleavage products, we have argued that the strategy must be to prevent the 2'OH at the cleavage site from acting as a nucleophile and instead facilitate nucleophilic attack from the other side of the phosphorous center to ensure correct cleavage products (5). Thus, positioning of metal ions in relation to the cleavage site is of fundamental importance to ensure correct cleavage and to suppress unwanted hydrolysis of the RNA.
Figure 1 (A) Structure of the different pATSer variants used. Y indicates U or C at this position, I = inosine, 2AP = 2-aminopurine, DAP = 2, 6-diaminopurine, Pu = purine, dG = deoxyG, ddG = c7deoxyG, Rib = ribavirin, 3 mU = 3-methyl U, denotes that the base at this position was deleted while cs and red arrow = canonical RNase P cleavage site between positions –1 and +1. Blue arrows indicate Pb2+-induced cleavage sites (note that Pb2+ also promoted cleavage at position +2, not indicated in the figure but see text) and in the case of the U–1 variants the blue arrows indicate that cleavage was observed at +1 in addition to cleavage at the other positions. The elips denote the residues that had been deleted in these variants. (B) The structures of guanosine (G; top) and uridine (U; bottom), the red circles indicate the chemical groups that were substituted while the grey area corresponds to the part of G that is missing in ribavirin, for details see text. (C) Model of the RCCA–RNase P RNA interaction (interacting residues underlined). The letters A–C refer to divalent metal ions that have been identified in the substrate and in the P15 loop (for references see text). The A248/N–1 interaction is indicated (D) Suggested mechanism for Me2+-induced e.g. Pb2+ or Mg2+ cleavage of RNA. The Me2+ acts as a general base and activates the 2'OH resulting in 5'OH and 2';3'P as cleavage products (left). In RNase P RNA mediated cleavage available data suggest that Me2+-OH acts as the nucleophile resulting in 5'P and 3'OH as cleavage products (right). Me encircled in red denotes the divalent metal ion.
RNase P RNA is the catalytic subunit of the endoribonuclease RNase P that is responsible for generating the mature 5' termini of tRNA (6,7). Cleavage requires the presence of divalent metal ions, with Mg2+ as the preferred divalent metal ion. However, the presence of other divalent metal ions such as Mn2+ and Ca2+ can also promote cleavage . Also, combinations of divalent metal ions that do not promote cleavage (or do so poorly) when present alone resulted in increased cleavage activity when present in combination, e.g. mixing Sr2+ with Mn2+ or Zn2+ (9,10). This suggests that there is metal ion cooperativity in RNase P RNA mediated cleavage (9). One of the metal ions involved in this cooperativity was suggested to be positioned in the vicinity of the interaction between the 3' end of the substrate and RNase P RNA, the RCCA–RNase P RNA interaction . According to our model, the other metal ion(s) would be positioned at or in the vicinity of the cleavage site and be involved in generating the nucleophile (Figure 1C). Recently, we presented functional and structural evidence that substitution of the base pair at the cleavage site (the +1/+72 bp) influenced Mg2+ binding in its vicinity (12). This finding is in keeping with previous data suggesting that a metal ion is bound in the vicinity of the cleavage site. Earlier data also suggest that a metal ion coordinated H2O acts as the nucleophile (5,13–24). In the present investigation, we were initially interested to study the structural requirements for metal ion binding at the RNase P cleavage site. Therefore, we studied Pb2+-induced cleavage of model RNA hairpin RNase P substrates carrying changes at the cleavage site. Moreover, based on our previous data demonstrating that Pb2+ promotes hydrolysis of model RNA hairpin substrates near the RNase P cleavage site in the absence of RNase P RNA (21) and that combinations of metal(II) ions that do not promote cleavage (or do so poorly) when present alone can result in increased cleavage activity, we were also interested to investigate whether Pb2+ in combination with other divalent metal ions can promote RNase P RNA mediated cleavage.
Here we present data suggesting that the presence of a purine at the +1 position in the substrate suppresses Pb2+-induced cleavage at this position. However, a guanosine at +1 resulted in M1 RNA (RNase P RNA derived from Escherichia coli) mediated cleavage at the correct position in the presence of Pb2+ and Sr2+ or as the only divalent metal ions. Thus, RNase P RNA has leadzyme properties. The observation that Sr2+ or did not promote cleavage alone is in keeping with a model that Pb2+ is responsible for activating the nucleophile and we identified residues in both RNase P RNA and in the substrate that influence cleavage in the presence of Pb2+. Our findings provide experimental evidence for the model suggesting that the establishment of the RCCA–RNase P RNA interaction results in a re-coordination of Mg2+ positioned in the vicinity of the cleavage site (25).
MATERIALS AND METHODS
Preparation of substrates and RNase P RNA
The various pATSer derivatives were purchased from Dharmacon, USA, purified, labeled at the 5' end and gel purified according to standard procedures as described elsewhere (11,26,27). The RNase P RNA variants were generated as run-off transcripts using T7 DNA-dependent RNA polymerase (27,28).
Assay conditions and determination of the kinetic constants under single turnover conditions
The assays were performed under single turnover conditions at pH 6.1 or 7.2 (as indicated) in Buffer B: 50 mM Bis-Tris Propane, 5% (w/v) PEG 6000, 100 mM NH4Cl and at different concentrations as indicated. All reactions were performed at 37°C. Reaction products were separated on denaturing 20–22% (w/v) polyacrylamide gels and quantified using Phosphorimager (Molecular Dynamics 400S) as described elsewhere (5). The final concentrations of RNase P RNA and substrate were <20 μM and 3.2 μM, respectively.
The kinetic constants kobs and kobs/Ksto (= kcat/Km) were determined under saturating single turnover conditions at pH 6.1 in Buffer B as described previously (5,12) at: = 40 mM; = 40 mM and = 8 mM; = 40 mM and = 8 mM; = 160 mM and = 8 mM. The final concentration of substrate was <20 nM whereas the concentration of RNase P RNA was varied between 0.025 and 6.4 μM dependent on substrate and RNase P RNA variant. For the calculations we used the 5' cleavage fragments and the time of cleavage was adjusted to ensure that we were in the linear part of the curve of kinetics. The kinetic constants were obtained by linear regression from Eadie–Hofstee plots.
Binding assay conditions
Spin columns were used to determine the apparent equilibrium dissociation constant (appKd) as described elsewhere (5,12) in Buffer C: 50 mM MES at pH 6.0, 0.8 M NH4OAc, 0.05 %(w/v) Nonidet P40, 0.1% (w/v) SDS and different concentration of CaCl2 or SrCl2 as indicated. The final concentration of substrate was <20 nM and was varied between 0.025 to 6.4 μM (concentration range depended on RNase P RNA variant). The appKd was determined by using OriginPro 7.0 software and the equation fc = ft x free/(Kd + free), where fc represent fraction of substrate in complex with RNase P RNA and ft = maximum fraction of substrate able to bind RNase P RNA, i.e. end point.
Lead(II)-induced cleavage conditions
The different 5' end labeled pATSer derivatives were subjected to Pb2+-induced cleavage in Buffer A: 50 mM Tris–HCl pH 7.2, 5% (w/v) PEG 6000, 100 mM NH4Cl, 40 mM MgCl2 (or SrCl2) and PbOAc at a final concentration of 4 mM. The substrates were preincubated in Buffer A in the absence of PbOAc for 10 min at 37°C, incubated for 5 min after addition of PbOAc and the cleavage products were analyzed by gel-electrophoresis as described elsewhere (21,27).
Determination of the site of cleavage
Cleavage at +1 was inferred by comparing the mobility of the 5' cleavage fragments generated in the presence of Pb2+/Sr2+ and Mg2+ (see above). To verify the presence of 5' phosphate at the 5' termini of the cleaved product (internally labeled with GTP), the large cleavage product was gel purified and subjected to digestion with RNase T1, RNase T2 and pancreatic RNase A as described previously (29). Thin-layer chromatography according to Nishimura (30) was used to detect the 5' phosphate-labeled nucleotide i.e. pGp.
RESULTS
A purine at +1 suppresses metal induced cleavage at this position
The pATSerCG substrate is a well-characterized RNase P substrate that is cleaved at several positions in the 5' leader in the presence of Pb2+ and absence of RNase P RNA (21). A similar type of substrate was also used elsewhere and it was demonstrated that Mg2+ induced cleavage in the 5' leader the vicinity of the RNase P cleavage site (15). Hence, to investigate the structural requirements for positioning of a metal(II) ion at the RNase P cleavage site, we introduced changes at positions –1, +1 and/or +72 in pATSer (Figure 1A; the residue that pairs with residue +1 corresponds to residue +72 in tRNA and will therefore be referred to residue +72). These pATSer variants were subjected to Pb2+-induced cleavage. As shown in Figure 2, introduction of a U or C at +1 resulted in significant cleavage at +1 irrespective of identity of the residue at +72 or –1. By contrast, a significant reduction in Pb2+-induced cleavage at +1 was observed due to the presence of purine derivatives at this position. Moreover, our data suggested that base pairing between U+1 and the base at +72 did not influence Pb2+-induced cleavage at +1 (Figure 2, compare cleavage of U+1/A+72, U+1/DAP+72, U+1/C+72 and 3mU+1/A+72 variants). Neither did substitution of N7 of G+1 with c7 as was evident when Pb2+ cleavage of pATSerUGG/C, pATSerUGdG/C and pATSerUG(c7dG)/C was compared (data not shown; underlined residues corresponds to residues G+1 and C+72) or in the absence of the 3' terminal sequence G+73CCAC (Figure 1A). A significant Mg2+-induced cleavage at +1 was also observed due to replacement of G+1/C+72 with U+1/A+72 (data not shown). Surprisingly, introduction of ribavirin at +1 resulted in a similar suppression of Pb2+-induced cleavage at +1 compared to when the substrate carried a purine at this position.
Figure 2 Histograms showing Pb2+-induced cleavage of the different substrates in the absence of RNase P RNA as indicated. The distribution of the frequency of cleavage at –1, +1 and +2 were calculated and the relative distribution in percent was plotted as indicated. gccac indicate residues that were deleted in these pATSerUG derivatives as illustrated in Figure 1.
The 5' cleavage products generated as a result of addition of Pb2+ migrated slightly slower compared to the migration of the 5' cleavage fragments produced in the M1 RNA mediated cleavage reaction (see below Figure 3A and data not shown). In addition, the Pb2+ induced 5' cleavage products could not be labeled at their 3' ends with pCp (data not shown). These findings are expected given that metal(II)-induced cleavage generates cleavage product ends with 5'OH and 2';3' cyclic phosphates (2,3). Analysis of the 5' end of the larger cleavage product by thin-layer chromatography (i.e. the presence of nucleotides with a 5' phophate could not be detected; Figure 3B upper panel) is in agreement with these findings.
Figure 3 (A) M1 RNA mediated cleavage of pATSerUGG/C, pATSerUGIno/C and pATSerUG2AP/U under single turnover conditions at pH 7.2 in the presence of 8 mM Pb2+ and 40 mM Sr2+ as indicated. Two different M1 RNA variants (3.2 μM final concentration; final concentration of substrate 0.04 μM), wild-type M1 RNA (the number 3 lanes) and M1C294 RNA (the number 4 lanes) were used. Two controls, cleavage of the substrates in the absence of M1 RNA and ± tRNA were included (no tRNA added the number 1 lanes while in the number 2 lanes tRNA was added). The filled circles indicate M1 RNA mediated cleavage at –1. The cleavage reaction was terminated after 5 min (for details see text). The different fragments as a result of cleavage at –2, –1 and +1 are shown. (B) Two-dimensional thin-layer chromatography according to Nishimura (30) demonstrating the presence of 5' pGp (in red) as a result of M1 RNA mediated cleavage in the presence of Pb2+. Internally labeled GTP pATSerUGG/C was incubated in the presence of Pb2+/Sr2+ and in the absence and in the presence of M1 RNA as indicated. The large cleavage product was isolated and subjected to RNase digestion as outlined in Materials and Methods. For further experimental details see text. (C) and (D) M1 RNA mediated cleavage expressed as rate of cleavage in % per min as a function of increasing concentration of Sr2+ (C) and Pb2+ (D) using wild-type M1 RNA and M1C294 RNA as indicated. When Sr2+ was increased the Pb2+ concentration was kept constant at 8 mM likewise when we varied the Pb2+ concentration the Sr2+ concentration was kept constant at 40 mM. The experiments were performed at pH 6.1 at 37°C and the concentrations of M1 RNA and substrate were 3.2 μM and 40 nM, respectively. (E) M1 RNA mediated cleavage expressed as relative cleavage at +1 in % as a function of increasing concentration of as indicated using wild-type M1 RNA. The Pb2+ concentration was 8 mM. The experiment was performed in Buffer B at pH 7.2 at 37°C and the concentrations of M1 RNA and substrate were 3.2 μM and 40 nM, respectively.
We conclude that the local structural architecture around the +1 residue influence the positioning of Pb2+ (or Mg2+) in its vicinity. More specifically we suggest that the presence of a purine at +1 prevents substantial metal induced cleavage at +1 and that the Hoogsteen surface of purine plays an important role. The observed cleavage at +1 for the U+1 and C+1 derivatives could be due to the electronegative environment generated by the oxygen at position 2 of U or C in the shallow groove (see also below). In all other cases tested there is either an exocyclic amine or no chemical group in the shallow groove at this position in the substrate.
RNase P RNA has leadzyme properties
The presence of Pb2+ induces hydrolysis of the phosphate backbone at the RNase P cleavage site (and in its vicinity) in the precursor substrate while combinations of metal ions that do not promote cleavage by themselves (or do so poorly) results in increased RNase P RNA mediated cleavage (9). Pb2+ was not included in our previous studies since we argued that addition of Pb2+ results in cleavage of RNase P RNA as well as other RNAs in particular at pH > 7 (2,3,9). Nonetheless, we decided to investigate whether RNase P RNA can mediate cleavage at the correct position in the presence of Pb2+. Conceivably this would give us a tool to identify factors important for generating 5'P and 3'OH as cleavage products on the one hand, and 5'OH and 2';3' cyclic phosphate on the other, where the former cleavage products are hallmarks for RNase P RNA promoted cleavage and the latter the signature for Me2+- (here Pb2+-) induced cleavage . Addition of Pb2+ will result in cleavage of both M1 RNA and the substrate pATSer at specific as well as unspecific positions (3,7). Thus, cleavage conditions had to be short to minimize RNA degradation by Pb2+ but long enough to be able to detect RNase P RNA mediated cleavage. To achieve this objective we used E.coli RNase P RNA, M1 RNA and pATSerUGG/C as substrate. The pATSerUGG/C substrate (referred to as ‘wild type’) has in our previous studies been demonstrated to be as efficiently cleaved as precursor tRNAs (5,11,12). We preincubated M1 RNA for 10 min at 37°C in the presence of various concentrations of Sr2+ to allow M1 RNA to fold into an active conformation. No difference in the conformation of M1 RNA due to replacement of Mg2+ with Sr2+ was observed using Pb2+-induced cleavage to monitor conformational changes (21). Note also that Sr2+ alone does not promote M1 RNA mediated cleavage under these conditions . The substrate was added (amount added to ensure single turnover conditions) and after an additional incubation for 5 min Pb2+ was added to a final concentration of 8 mM (or as indicated). The cleavage reaction was terminated after 5 min and the cleavage products were separated by gel-electrophoresis (see Materials and Methods). Surprisingly, we observed M1 RNA mediated cleavage at the correct position +1 under these conditions with an optimum cleavage rate at 8 mM Pb2+ and 40 mM Sr2+ (Figure 3A). That cleavage was indeed mediated by M1 RNA was confirmed by employing thin-layer chromatography to demonstrate the presence of pGp at the 5' end of the cleavage product (Figure 3B). We also tried the combination Pb2+ and . Here we had to reduce the concentration of significantly to detect M1 RNA mediated cleavage since higher concentrations of resulted in inhibition (Figure 3E). The lower concentration of is rationalized by that binds to RNA with higher affinity compared to e.g. Sr2+ (21). Consequently, at higher concentration of , most likely displace Pb2+ and thereby cleavage is inhibited (see also below). Taken together, these findings are in keeping with our study where we observed cleavage when we mixed Sr2+ or with Zn2+ (9). In this context we note that Taira and coworkers (4) have studied the hammerhead cleavage reaction in the presence of different metal ions. Their data suggest that La3+ can act both as an activator and inhibitor of cleavage .
A possibility is that the observed activities are due to contamination of Mg2+ or Ca2+. However, in the presence of 40 mM Sr2+ and 0.1 mM Mg2+ we did not observe M1 RNA mediated cleavage (data not shown). Also, cleavage in the presence of 15 mM Ca2+ and 25 mM Sr2+ reduce the efficiency of cleavage >100-fold compared to cleavage in the presence of 15 mM Mg2+ and 25 mM Sr2+ (9). Hence, since addition of 0.1 mM Mg2+ did not result in cleavage we do not expect that addition of Ca2+ would. These data argue against contamination of Mg2+ and Ca2+.
Replacing pATSerUGG/C with a conventional tRNA precursor, pSu3, also resulted in cleavage at the correct position at +1 in the presence of Pb2+ and Sr2+ (data not shown). Finally, Pb2+ induced cleavage of 32P-labeled M1 RNA showed very little degradation of M1 RNA after 5 min in the presence of Pb2+ (data not shown).
We conclude that M1 RNA mediated cleavage can be promoted in the presence of Pb2+ and Sr2+ (or ), where Sr2+ and do not promote cleavage when present alone. Thus, M1 RNA has leadzyme properties. This finding is in keeping with a model that Pb2+ is involved in generating the nucleophile i.e. corresponding to MeA in Figure 1C. Given this, the MeB binding site in the M1 RNA substrate (RS-) complex would most likely be occupied by Sr2+ (or ) since a Pb2+ at this position would result in hydrolysis of the M1 RNA phosphate backbone in its vicinity .
The leadzyme-like activity is not a unique feature of M1 RNA
To investigate whether leadzyme-like activity could be observed for other RNase P RNAs we replaced M1 RNA with RNase P RNA derived from Mycoplasma hyopneumoniae, Hyo P RNA (32) and Yersinia pestis, Yer P RNA (Figure 4; B. M. F. Pettersson and L. A. Kirsebom, unpublished data). The former is a type B RNase P RNA and the latter is closely related to M1 RNA i.e. type A. While Hyo P RNA did not mediate cleavage under these conditions, partly due to poor substrate binding (data not shown), Yer P RNA promoted cleavage of pATSerUGG/C at the correct position +1 in the presence of Pb2+/Sr2+ although less efficiently compared to M1 RNA (Figure 5). Both Hyo P RNA and Yer P RNA cleaved pATSerUGG/C efficiently and correctly in the Mg2+ alone cleavage reaction i.e. 40 mM Mg2+ (Figure 5; data not shown for Hyo P RNA). Moreover, the appKd value for Yer P RNA indicated no apparent difference in substrate binding compared to M1 RNA (see below; Table 1). Thus, the leadzyme-like activity is not a unique feature of M1 RNA.
Figure 4 The predicted secondary structure of Yersinia pestis RNase P RNA according to Brown (48). The positions/region of the different changes that were introduced are highlighted in red.
Figure 5 Cleavage rates under single turnover conditions at pH 6.1 as expressed in percentage of cleavage as a function of time for the different RNase P RNA variants as indicated where Yer P represents RNase P RNA derived from Yersinia pestis. The upper panel shows cleavage in the presence of 8 mM Pb2+ and 40 mM Sr2+ and the lower panel in the presence of 40 mM Mg2+. The concentrations of M1 RNA and substrate were 3.2 μM and 40 nM, respectively.
Table 1 Summary of the kinetic constants kobs and kobs/Ksto, and the apparent binding constants, appKd for various RNase P RNAs and pATSerUGG/C at Me2+ concentrations as indicated
Determinations of the apparent binding constant, appKd and the rate constant kobs
Next we determined the apparent binding constant, appKd, at two different Sr2+ concentrations 40 and 160 mM at pH 6.0. This was followed by determinations of the rate constant kobs under single saturating conditions at pH 6.1. At this pH the rate of cleavage is linearly dependent on in the presence of 8 mM Pb2+ and 40 (or 160 mM) mM Sr2+ suggesting that chemistry is rate-limiting at this pH . The linear dependence on is also consistent with that a hydroxyl ion is acting as the nucleophile . To be able to evaluate Pb2+ as a promoter of RNase P RNA mediated cleavage we also determined kobs in the presence of 8 mM Mg2+ and 40 mM Sr2+ at pH 6.1. The results are shown in Table 1.
Comparing appKd for wild-type M1 RNA determined in the presence of Ca2+ or Sr2+ indicated that pATSerUGG/C binds with 10- and 2.5-fold reduced affinity at 40 or 160 mM Me2+, respectively, in the presence of Sr2+. The rate of cleavage at position +1 in the presence of Pb2+ was only reduced 6-fold compared to cleavage in the presence of Mg2+ under the same conditions indicating that Pb2+ promoted cleavage with reasonable rates. Moreover, increasing the Sr2+ concentration resulted in better binding (20-fold) of the substrate but an almost 10-fold reduction in the rate of cleavage. Based on the model that Pb2+ generates the nucleophile this suggested that Sr2+ presumably had replaced Pb2+ in the vicinity of the cleavage site i.e. MeA (Figure 1).
Structural architecture of the cleavage site affects positioning of Pb2+
The observation that Pb2+ is positioned close to the cleavage site and likely involved in generating the nucleophile gives us a handle to identify residues and chemical groups in M1 RNA as well as in the substrate influencing RNase P RNA mediated cleavage promoted by Pb2+ (and most likely Mg2+). To address which chemical groups at the cleavage site are important for efficient cleavage in the presence of Pb2+, we cleaved pATSer variants with inosine (and C+72) and 2-aminopurine (and U+72) at +1, respectively (Figure 1A). Here we observed that cleavage of the ‘inosine’ variant was significantly reduced while the level of cleavage of the latter was not significantly different compared to cleavage of the ‘wild-type’ substrate, pATSerUGG/C (Figure 3A). This suggested that the exocyclic amine at position 2 in guanosine plays an important role possibly influencing positioning of Pb2+. The importance of the exocylic amine at this position is consistent with previous findings . Note also that we observed M1 RNA mediated cleavage at –1 in particular using M1C294 RNA (see below). We also tried to cleave the substrate where G+1/C+72 had been replaced with U+1/A+72 in the presence of Pb2+/Sr2+ but no cleavage was observed under these conditions most likely due to this substrate being a poor substrate for M1 RNA mainly due to its influence on metal ion binding in its vicinity . These data suggest that the structural environment at the cleavage site and in particular the exocyclic amine positioned in the shallow groove influenced Pb2+/Sr2+ promoted cleavage. We argue that these results are due to a change in the positioning of the Pb2+ involved in activating the nucleophile.
Structural architecture of ‘the +73/294 interaction’ affects positioning of Pb2+
We recently provided experimental evidence that there is cross talk between ‘the +73/294 interaction’ and the cleavage site. During this process metal ions are suggested to play important roles . Hence, we asked whether a change in the structural architecture of the +73/294 interaction influences the rate of cleavage in the Pb2+-promoted reaction. Based on our model that Pb2+ is located close to the cleavage site (see above) we reasoned that this would give information about whether the positioning of this Pb2+ ion(s) is affected or not. Thus, we used a mutant M1 RNA where U294 had been replaced with a C, M1C294 RNA, resulting in a G+73/C294 interaction instead of a G+73/U294 in the RS-complex (Figure 1B). The ground state binding (appKd at 40 mM Ca2+) of this mutant is very similar compared to the wild-type (Table 1). Moreover, as shown in Table 1 in the Mg2+ alone reaction the rate of cleavage of pATSerUGG/C was only modestly reduced in keeping with our previous data (5). By contrast in the presence of Sr2+, appKd was determined to be 6.5- to 2.5-fold lower relative to wild-type at 40 and 160 mM Sr2+, respectively, indicating an improved ground state binding for this mutant in the presence of Sr2+. Cleavage under single turnover conditions as a function of revealed that the C294 mutant cleaved the substrate more efficiently compared to the wild-type. This was observed to be the case irrespective of whether Mg2+ or Pb2+ promoted cleavage was studied (Figure 4; data only shown for the case when 8 mM Pb2+ was used). This was further corroborated when we determined kobs for M1C294 RNA under the conditions indicated in Table 1. Here we observed a 2.6-fold increase at 40 mM Sr2+/8 mM Mg2+ and a 4.1-fold increase at 40 mM Sr2+/8 mM Pb2+. At 160 mM Sr2+/8 mM Pb2+, we observed only a 1.8-fold increase relative to wild-type again indicating (see above) that higher concentration of Sr2+ inhibits the rate of cleavage under these conditions. These findings suggested that changes of the structural architecture of the +73/294 interaction influence metal ion binding most likely in its vicinity (MeA in Figure 1C; see also below). We argue that as a consequence, this affects the rate of cleavage possibly due to a repositioning of the metal ion involved in generating the nucleophile. This further indicates the role of metal ions bound at different sites in RNase P RNA mediated cleavage .
Identification of a residue in RNase P RNA affecting positioning of Pb2+
The efficiency of the Pb2+/Sr2+ promoted cleavage for Yer P RNA was lower compared to that of M1 RNA. The secondary structures of these two RNase P RNAs are very similar and differences were identified in four regions: C125A, U333G, U364C (residue in M1 RNA underlined) and AUAA in M1 RNA (see Figure 4) while Yer P RNA carries GCAU at this site. This gave us a way to identify other residues/regions in RNase P RNA potentially involved in positioning Pb2+ in the vicinity of the cleavage site. Thus, we generated four Yer P RNA variants carrying C125, U333, U364 and AUAA269 (M1 RNA numbering; Figure 4) to investigate whether the Yer P RNA activity could be rescued. As shown in Figure 5, only the C to U replacement at 364 resulted in an increased cleavage activity while the other changes did not. This was observed both in the Pb2+/Sr2+ and Mg2+ promoted reactions. Neither of these changes affected ground state binding (Table 1). These findings raise the possibility that the residue at 364 in Yer P RNA influenced positioning of the Pb2+ in the vicinity of the cleavage site.
DISCUSSION
Lead(II) ion is an efficient inducer of hydrolysis of the phosphate backbone of RNA generating 5'OH and 2';3'P as cleavage products (2,3). Here we showed that RNase P RNA is able to cleave its substrate at the canonical site in the presence of the Pb2+ and Sr2+ or .
The pKa value for Sr2+ is significantly higher compared to Mg2+ and Ca2+ that do promote RNase P RNA mediated cleavage (2,7). In a previous study Sr2+ was demonstrated to promote cleavage at elevated pH, pH 9 (14). Thus, one could argue that Pb2+ binding in the RS-complex ‘activates’ Sr2+ that subsequently results in generation of the nucleophile. Here we observed RNase P RNA mediated cleavage in the presence of Pb2+/Sr2+ at a significantly lower pH (6.1 and 7.2). When present alone, Sr2+ (or ) has not been demonstrated to promote cleavage by RNase P RNA under these conditions, i.e. at pH 7.5 . Also, an increase in the Sr2+ concentration resulted in inhibition of cleavage. Moreover, mixing Pb2+ with resulted in RNase P RNA mediated cleavage and increasing concentration of also inhibited cleavage. Together this argues against ‘activation’ of Sr2+. Rather the role of Sr2+ (or ) is suggested to promote RNA folding and facilitate/stabilize the interaction with the substrate. Note that available data suggest that RNase P RNA is not correctly folded as well as that it is efficiently cleaved in the presence of only Pb2+ (21). Our data also argues against the presence of contamination of e.g. Mg2+ in our solutions. We conclude that our data are consistent with a model where Pb2+ most likely is responsible for generating the nucleophile. To conclusively demonstrate that this is indeed the case will require detailed structural analysis of the RNase P RNA substrate complex.
Compared to Mg2+ that is the preferred metal(II) ion in promotion of RNase P RNA mediated cleavage, Pb2+ shows different physico-chemical properties but their preferred coordination numbers are similar: Pb2+ 6 to 8 and Mg2+ 6 (36). Moreover, although the softer Pb2+ ion is larger than Mg2+, Pb2+ and Mg2+ bind to overlapping sites (2). This, together with that Pb2+ can act as an efficient general base (pKa 7.8) as well as the similarity in coordination numbers might be the only reasons to why Pb2+ can promote RNase P RNA mediated cleavage. Whether other criteria also has to be fulfilled remains to be elucidated. But, unlike lead(II)-induced hydrolysis or that by the in vitro evolved leadzyme which generate 5'OH and 2';3'P as cleavage products (37), Pb2+-promoted cleavage by RNase P RNA resulted in 5'P and 3'OH. Therefore, the mechanism of cleavage has to be different and, in the case of RNase P RNA the nucleophilic attack has to come from the opposite side relative to the positioning of the 2'OH at the cleavage site (Figure 1C). Available data suggest that H2O is the nucleophile in RNase P RNA mediated cleavage (13,17,24). In keeping with this we demonstrated that cleavage by RNase P RNA in the presence of Pb2+/Sr2+ is linearly dependent on (see above). Thus, most likely a H2O ligand is also the nucleophile in RNase P RNA cleavage in the presence of Pb2+ i.e. Pb2+ acts as a general base. To our knowledge this is the first time that Pb2+ has been suggested to activate H2O to act as a nucleophile in RNA mediated cleavage of RNA. To conclude, our present finding that Pb2+ likely act as a catalytic metal ion in M1 RNA mediated cleavage gives us a handle to identify factors/chemical groups that promote M1 RNA mediated cleavage and suppress cleavage of the phosphate backbone in the 5' leader due to activation of neighboring 2'OH groups and thereby increase our understanding of cleavage of RNA by RNA.
Close and distant residues affect positioning of Pb2+ in the RS-complex
Based on the discussion above Pb2+ promotes cleavage at and in the vicinity of the RNase P cleavage site both in the absence and presence of RNase P RNA (Figure 3), where efficient cleavage at the canonical site (with correct cleavage products and G at +1) was only observed in its presence. Hence, it is conceivable that Pb2+ positioned in the vicinity of the cleavage site is re-positioned as a result of formation of the RS-complex arguing for the possibility that the metal ion responsible for generating the nucleophile is associated with the substrate . The importance of the structural architecture of the +1/+72 for metal ion binding in the vicinity of the cleavage site as well as the finding that the presence of the exocyclic amine at position 2 of the guanosine at +1 is important for M1 RNA mediated cleavage in the presence of Pb2+ is in keeping with this model . However, we cannot exclude the possibility that the metal(II) ion(s) involved in generating the nucleophile is recruited from the solution or is associated with RNase P RNA e.g. Me2+ bound in the P4 helix (8).
Irrespective of whether MeB (Figure 1C) is associated with the substrate, RNase P RNA or is recruited from the solution, our data indicate that the structural architecture of the +73/294 interaction influenced the rate of cleavage promoted in the presence of Pb2+ as well as metal(II) binding in its vicinity. This is consistent with our previous findings where we showed that the structural architecture of the +73/294 interaction, that is part of the RCCA–RNase P RNA interaction (25), affects the charge distribution at the cleavage site, ground state binding, rate of cleavage and metal ion binding in its vicinity . Thus, our present findings provide further evidence for cross talk between the +73/294 interaction and the site of cleavage and experimental evidence for the involvement of metal ion(s) in this process.
Our data also indicate that U364 in M1 RNA is likely to influence positioning of Pb2+ in the vicinity of the cleavage site. The significance of this finding is not clear yet but it might indicate that either that this region is positioned in the vicinity of the cleavage site or that this change results in distance effects e.g. by affecting positioning of the metal(II) ions positioned in the P4 helix . However, based on the recent RNase P RNA crystal structures the former alternative is less likely (39,40). Clearly we need to investigate this in further detail but nonetheless this finding opens for new possibilities and approaches using hybrid RNase P RNA molecules in our efforts to understand the complex orchestration in RNase P RNA mediated cleavage. In this context we note that the classical double mutant M1 RNA ts709 carries a G to A change at 365. However, no phenotype has been linked to this change [(41–43); L. A. Kirsebom and S. Altman, unpublished data). Thus, our findings raise the possibility that this mutation indeed influences M1 RNA mediated cleavage by affecting positioning of the metal ion(s) involved in activating the nucleophile.
Structural evidence for positioning a metal(II) ion at the RNase P cleavage site
Our present and previous data clearly indicate that a divalent metal ion(s) is positioned close to the RNase P cleavage site (5,13–24). The high-resolution structure of the RNase P cleavage site is not yet available. However, we noticed that the structures of the ends of the leadzyme are almost identical to the sequences at and in the vicinity of the pATSerCGG/C and pATSerCGU/A cleavage sites with the exception that in the case of the ‘G+1/C+72’ variant the C–1 is single stranded . But in another high-resolution structure of SRP RNA in which the structure at one of the ends is also identical to the sequence of the pATSerCGG/C cleavage site the C–1 is base paired to a G as in pATSerCGG/C . These structural studies suggested that a hexahydrated Mg2+ is bound in the deep groove in the vicinity of the base pairs that would correspond to G+1/C+72 and U+1/A+72, respectively, in our substrates. Superimposing the three Mg2+ binding sites revealed no apparent structural differences. But, the positioning of Mg2+ is suggested to be shifted >7 ? due to the G+1/C+72 to U+1/A+72 replacement, while formation of the ‘C–1/G+73’ base pair only gives a shift of 2.2 ?. In our substrates this would correspond to the Mg2+ near the cleavage site. This supports our functional data suggesting that U/A at +1/+72 affects Pb2+ as well as Mg2+ binding most likely in the vicinity of the cleavage site . Hence, we suggest that there is a functionally important Mg2+, most likely in the hexahydrated state, positioned in the deep groove at the cleavage site. Based on this model it is apparent that hexahydrated Mg2+ is positioned close to the cleavage site (7–8 ?) and to the phosphorous centers of +1, –1 and –2 (Figure 6). Assuming that this is the metal ion that is involved in generating the nucleophile (see above) a repositioning has to occur as a result of the interaction with RNase P RNA to ensure cleavage at +1 i.e. positioning of the nucleophile for an in line attack on the +1 phosphorous center. During this conformational rearrangement, the data suggest that the ‘73/294 interaction’, residue 364 and the exocyclic amine of G+1 in the substrate play important roles (see above). At the same time, attack on the phosphorous center at –1 and –2 should be avoided. In the case of cleavage in the presence of Pb2+ this is not observed due to that Pb2+ is very efficient to activate the neighboring 2'OH to act as a nucleophile while Mg2+ is significantly less efficient (2). This rationalizes why cleavage at other positions within the 5' leader under Pb2+/Sr2+ conditions even in the presence of M1 RNA was observed.
Figure 6 The three dimensional structures derived from the RNA leadzyme (43) and SRP RNA (44) superimposed where the upper representation is viewed from the side and the lower from the top. Two structures, I and III, were taken from the RNA leadzyme (to the left in the figure) whereas structure II was derived from the SRP RNA. The hexahydrated Mg2+ ions are designed B1, B2 and B3 where B1 corresponds to the Mg2+ located in the SRP RNA (II), while B2 and B3 are the Mg2+ ions located in the vicinity of ‘G+1C+72’ (structure I) and ‘U+1A+72’ (III) variants in the RNA leadzyme, respectively. The dashed arrows in parenthesis in the left half of the figure indicate the sites that correspond to the canonical RNase P cleavage site. The red arrows mark the phosphorous atom that corresponds to the one to be attacked in RNase P RNA mediated cleavage. The residues that would correspond to C–1 and G+73 in our pATSer substrate derivatives are indicated in the three dimensional structure representations.
Concluding remarks
RNase P RNA is the first natural ribozyme demonstrated to show leadzyme-like activity. Hence, our findings raise the question whether other ribozymes can mediate catalysis using Pb2+. In this context we note that Mg2+ and Mn2+ promote correct splicing while Mn2+ also induce cleavage at an additional site in the Group I intron. However, Pb2+ does not promote splicing but induce cleavage at the additional site due to that Pb2+ do not promote correct folding (46,47). In view of our present findings it would be of interest to test whether Pb2+ in combination with other divalent metal ions e.g. Sr2+ can promote Group I intron splicing. To the best of our knowledge this has not been tested.
ACKNOWLEDGEMENTS
We thank our colleagues for discussions in particular Drs F. Darfeuille and M. Br?nnvall. Drs S. Dasgupta, D. Hughes and A. Virtanen are acknowledged for critical reading of the manuscript. Finally we thank Ms U. Lustig for technical assistance. This work was supported by a grant to LAK from the Swedish Natural Research Council. Funding to pay the Open Access publication charges for this article was provided by Swedish Natural Research Council.
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