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Originally published In Press as doi:10.1074/jbc.M305939200 on August 6, 2003

J. Biol. Chem., Vol. 278, Issue 44, 43394-43401, October 31, 2003
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What's this?

Catalysis by RNase P RNA

UNIQUE FEATURES AND UNPRECEDENTED ACTIVE SITE PLASTICITY*

Tina Persson{ddagger}§, Simona Cuzic{ddagger}, and Roland K. Hartmann{ddagger}||

From the {ddagger}Universität zu Lübeck, Institut für Biochemie, Ratzeburger Allee 160, D-23538 Lübeck, Germany

Received for publication, June 5, 2003 , and in revised form, August 5, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Metal ions are essential cofactors for precursor tRNA (ptRNA) processing by bacterial RNase P. The ribose 2'-OH at nucleotide (nt) –1 of ptRNAs is known to contribute to positioning of catalytic Me2+. To investigate the catalytic process, we used ptRNAs with single 2'-deoxy (2'-H), 2'-amino (2'-N), or 2'-fluoro (2'-F) modifications at the cleavage site (nt –1). 2' modifications had small (2.4–7.7-fold) effects on ptRNA binding to E. coli RNase P RNA in the ground state, decreasing substrate affinity in the order 2'-OH > 2'-F > 2'-N > 2'-H. Effects on the rate of the chemical step (about 10-fold for 2'-F, almost 150-fold for 2'-H and 2'-N) were much stronger, and, except for the 2'-N modification, resembled strikingly those observed in the Tetrahymena ribozyme-catalyzed reaction at corresponding position. Mn2+ rescued cleavage of the 2'-N but also the 2'-H-modified ptRNA, arguing against a direct metal ion coordination at this location. Miscleavage between nt –1 and –2 was observed for the 2'-N-ptRNA at low pH (further influenced by the base identities at nt –1 and +73), suggesting repulsion of a catalytic metal ion due to protonation of the amino group. Effects caused by the 2'-N modification at nt –1 of the substrate allowed us to substantiate a mechanistic difference in phosphodiester hydrolysis catalyzed by Escherichia coli RNase P RNA and the Tetrahymena ribozyme: a metal ion binds next to the 2' substituent at nt –1 in the reaction catalyzed by RNase P RNA, but not at the corresponding location in the Tetrahymena ribozyme reaction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Endonucleolytic 5' maturation of tRNA primary transcripts is catalyzed by the ribonucleoprotein enzyme ribonuclease P (RNase P)1 in all three domains of life (Archaea, Bacteria, and Eukarya) as well as in mitochondria and chloroplasts (1–3). Bacterial RNase P enzymes are composed of a catalytic RNA subunit (4), ~400 nucleotides (nt) in length, and a single small protein of typically 120 amino acids (5). The only known exception may be the hyperthermophilic bacterium Aquifex aeolicus. Neither genes for RNase P RNA (rnpB) or protein (rnpA) subunits could be identified in the sequenced genome, nor have biochemical studies provided evidence for the presence of a bacterial-like RNase P activity (6).

Processing of ptRNAs by RNase P results in cleavage products with 3'-OH and 5'-phosphate termini. Studies with the model RNase P RNA ribozymes from Escherichia coli and Bacillus subtilis have indicated that at least two metal ions (the actual number influenced by the nature and concentration of monovalent cations present) play a specific role in productive enzyme-substrate complex formation and cleavage chemistry (7–14). A solvent hydroxide or activated water molecule is assumed to be positioned by a metal ion for nucleophilic attack in an SN2 in-line displacement mechanism (7, 15). A 2'-deoxyribose substitution at the RNase P cleavage site was previously reported to affect binding of catalytically important Mg2+ and to substantially reduce the rate of catalysis by E. coli RNase P RNA (7, 16–18). 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 "prebound" to ptRNA before complexation with RNase P RNA (19).

Here we have analyzed the role of the 2'-OH group at nt –1 of ptRNA substrates for cleavage by E. coli RNase P RNA (M1 RNA) in more detail by introducing a single 2'-amino (2'-N), 2'-fluoro (2'-F), or 2'-deoxy (2'-H) substitution at this position. We demonstrate that the 2'-OH at nt –1 of the substrate contributes to enzyme-substrate binding in the ground state and to transition state stabilization in particular. 10-fold slower cleavage chemistry of the 2'-F-ptRNA compared with the all-ribose control but a 13-fold rate advantage over the 2'-H-ptRNA suggests a hydrogen-bonding function for the 2'-OH at nt –1 during catalysis. All three 2' modifications at nt –1 caused a defect in the binding of catalytically relevant Mg2+. Increased miscleavage at position –2/–1 of 2'-N-modified substrate variants at lower pH suggests that the 2'-amino group becomes protonated and promotes electrostatic repulsion of the Me2+ bound in its immediate vicinity. The extent of miscleavage and the pH value at which we observed 50% miscleavage showed a remarkable dependence on the base identities at nt –1 and +73 of ptRNA. We further show that the replacement of Mg2+ with Mn2+ strongly improves processing efficiency for substrates with a 2'-N substitution at nt –1, although there is no convincing evidence for a direct metal ion coordination to the 2' substituent at nt –1.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
RNA Synthesis and 5' 32P-End-labeling—RNA oligonucleotides with and without single 2'-ribose modifications were either synthesized by phosphoramidite chemistry as described (20) or purchased from IBA (Göttingen, Germany). E. coli M1 RNA and the 3'-portion of ptRNAGly missing the first 17 nt of the mature domain (termed tRNA18–79; see also Fig. 1) were synthesized by T7 transcription; the latter as part of a precursor transcript flanked by self-cleaving 5'- and 3'-cis-hammerheads to generate homogeneous 5'- and 3'-ends. 5'-end-labeling was performed as described (21). RNAs were purified by denaturing polyacrylamide electrophoresis.



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  		FIG. 1.
ptRNAGly variants carrying single 2'-ribose modifications at the canonical RNase P cleavage site at nt –1. The arrow marks the canonical RNase P cleavage site between nt –1 and +1. Circled nt 17, 18 mark the site of ligation by T4 RNA ligase. Variants with C ->t U and/or U -> A exchanges at positions –1 and +73, respectively, are highlighted.

 

Alkaline and Nuclease P1 Hydrolysis—Alkaline and nuclease P1 hydrolysis assays were performed essentially as described (22)

Assembly of ptRNA Variants with Single Site Modifications—4.5 nmol of modified or unmodified RNA oligonucleotide (24 nt), covering the 7-nt 5'-flank and the first 17 nt of the mature domain of ptRNAGly (Fig. 1), 3 nmol of 5'-phosphorylated tRNA18- 79, 20 µl of 1 M HEPES (pH 7.5), and 20 µl of 0.1 M dithiothreitol were combined and adjusted with H2O to a final volume of 160 µl. The mixture was heated for 3 min at 90 °C, followed by incubation for 10 min at 65 °C and slow cooling (45 min) to ambient temperature in a metal block removed from the heating apparatus. The solution was then adjusted to 15 mM MgCl2, 0.5 mM ATP, 10% (CH3)2SO, and 140 units of T4 RNA ligase (Invitrogen) in a final volume of 200 µl, and incubated overnight at 16 °C. The sample and a 100-µl wash of the reaction tube were combined, and RNA was concentrated by ethanol precipitation overnight at –20 °C in the presence of 20 µg of glycogen as carrier. RNA pellets were redissolved in 15 µl of H20. Three samples prepared in this way were combined ({Sigma}45 µl), mixed with loading buffer (45 µl; Ref. 23) and loaded onto a 10% PAA/8 M urea gel (gel thickness: 1 mm; pocket width: 20 mm). Ligated pRNAs were detected by UV shadowing, excised from gels, and eluted from crushed gel slices in 200 mM Tris/HCl, 1 mM EDTA (pH 7.0) at 4–10 °C overnight, followed by a second elution for several hours. Eluted ptRNAs were concentrated by ethanol precipitation and redissolved in 50 µl of H2O.

Kinetics—Standard processing assays were performed under single turnover conditions (5 µM M1 RNA, < 1 nM ptRNA, 1 M NH4OAc, 50 mM MES, or HEPES; metal ion concentration and pH as indicated) as described (21). Aliquots withdrawn from enzyme-substrate mixtures were desalted by ethanol precipitation in the presence of 20 µg of glycogen before analysis by 20% PAGE/8 M urea. Data analysis and calculation of single turnover rates of cleavage (kobs) was performed as described (21).

The calculation of error bars in Fig. 3 is illustrated with the following representative example: the kobs values for all-ribose ptRNAGlyCU and ptRNAGlyCHU at pH 5.5 and 0.2 M Mg2+ were 5 ± 0.6 and 0.05 ± 0.015 min–1, respectively (each value based on five individual experiments); krel (OH/H) was calculated as 5/0.05 = 100; the upper and lower limits for krel were calculated as 5.6/0.035 = 160 and 4.4/0.065 = 68, respectively; thus, krel is illustrated as 100 with errors of +60 and –32.



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  		FIG. 3.
Secondary plots based on the data shown in Fig. 2A (A) and Fig. 2C (B and C). krel is the quotient of kobs for all-ribose ptRNAGlyCU divided by kobs for the respective modified substrate at the corresponding Mg2+ concentration: kobs OH/kobs F (•), kobs OH/kobs N ({blacktriangledown} or {blacktriangleup}), kobs OH/kobs H ({square}). For determination of error bars, see "Experimental Procedures." The data points for krel OH/N ({blacktriangleup}, panel C) are shown separately to avoid commingling of error bars with those for krel OH/H ({square}) in panel B.

 



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  		FIG. 2.
Processing of ptRNAGlyCU (•), CFU ({square}4), CNU ({circ}), and CHU ({blacktriangledown}) by E. coli M1 RNA under single turnover conditions at saturating enzyme concentrations (5 µM RNase P RNA, < 1 nM ptRNA, 1 M NH4OAc, 50 mM MES pH 5.5, or HEPES pH 7.0) and different divalent metal ion concentrations. A, observed single turnover cleavage rates (kobs) at pH 7.0 depending on the Mg2+ concentration. B, cleavage rates at pH 7.0 as in A but at two different Mn2+ concentrations. C, Mg2+-dependent cleavage rates at pH 5.5. D as C but omitting the curve for all-ribose ptRNAGlyCU to magnify the differences among the modified substrate variants.

 
Spin Column Assays—Spin column assays for the determination of apparent equilibrium dissociation constants (appKD) of enzyme-substrate complexes were performed as described (9, 24) in a buffer containing 50 mM MES pH 6.0, 1 M NH4OAc, 15 mM Ca(OAc)2, 0.1% (w/v) SDS, and 0.05 (w/v) Nonidet P40.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
ptRNA Ground State Binding to M1 RNA—Variants of the standard substrate ptRNAGlyCU (C-1, U+73) with a 2'-OH, 2'-F, 2'-N or 2'-H substituent at position –1 (referred to as ptRNAGlyCU, CFU, CNU, and CHU in the following; Fig. 1) were analyzed for binding to M1 RNA in the ground state. The 2' modifications had moderate (2.4–7.7-fold) effects on ptRNA binding, decreasing substrate affinity in the order 2'-OH > 2'-F > 2'-N > 2'-H (Table I). This was measured at pH 6.0 in the presence of 15 mM Ca2+ as the divalent metal ion to prevent substantial ptRNA cleavage during binding assays.


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  		TABLE I
Substrate ground state binding to E. coli RNase P RNA

Spin column assays were performed at 1 M NH4OAc, 15 mM Ca2+, pH 6.0. The appKd is apparent Kd. Results are based on three or more independent experiments. For calculation of {Delta}G, see Ref. 28.

 

Kinetics—The same substrate variants mentioned above were processed under single turnover conditions (see "Experimental Procedures") at saturating enzyme and varying divalent metal ion concentrations, either at pH 7.0 or pH 5.5. The pH of 7.0 was chosen to provide conditions where the 2'-amino group is mainly deprotonated. The pKa of the 2'-amino group was determined to be 6.2 (25) and 6.0 (26) in the context of dinucleotides. The pH 5.5 allowed us to analyze cleavage at rate-limiting chemistry for all substrates, including the most efficiently cleaved all-ribose substrate. Rate-limiting chemistry for the latter was inferred from the linear relationship of log kobs over pH in the range of pH 5.0–6.5 (8). For substrates with inherently slower cleavage chemistry, such as ptRNA with a 2'-deoxy modification at nt –1, the linear relationship of log kobs over pH persisted even up to pH values of about 8.0 in keeping with previous findings (Ref. 27; see also Fig. 5).



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  		FIG. 5.
A, pH-dependent processing of ptRNAGlyCNU by M1 RNA under single turnover conditions (1 µM RNase P RNA, <1 nM ptRNA, 1 M NH4OAc, 100 mM Mg2+, and either 50 mM MES pH 5.0 or 6.0, 50 mM PIPES pH 7.0 or 50 mM HEPES pH 8.0). B, linear relationship of kobs (logarithmic scale) and pH with a slope of 1.1; kobs values represent the sum of partial rates for cleavage at nt –1/+1 and –2/–1.

 

Cleavage efficiency of all-ribose and 2'-modified ptRNAGly CU variants at pH 7.0 decreased in the order 2'-OH > 2'-F > 2'-N > 2'-H (Fig. 2A). The same hierarchy was seen when Mg2+ was replaced with Mn2+ (Fig. 2B). A similar relationship was evident at pH 5.5 (Fig. 2, C and D), although the ptRNAGlyCNU was as inefficiently cleaved as the ptRNAGlyCHU, suggesting that protonation of the 2'-amino group at pH 5.5 impaired cleavage efficiency (see below). In Fig. 3, we plotted the data of Fig. 2, A and C as relative cleavage rates (krel) versus Mg2+ concentration. The krel values for processing at pH 7.0 (Fig. 3A) decreased with increasing [Mg2+], but remained rather constant between 0.04 and 0.1 M Mg2+. At 0.06 M Mg2+, the cleavage rate of the unmodified substrate was ~3-fold, 16-fold, and almost 40-fold higher than that of ptRNAGlyCFU, CNU and CHU, respectively. At pH 5.5 (Fig. 3, B and C), where the chemical step is rate-limiting, a linear relationship of krel versus [Mg2+] is clearly evident at [Mg2+] >= 0.1 M, with the rate for the unmodified ptRNA exceeding that of the modified substrates by a factor of ~10 (2'-F) and about 150 (2'-H, 2'-N). We are aware that the unphysiologically high concentrations of Mg2+ used here (up to 0.6 M) may have affected the conformational equilibria of substrate and ribozyme RNAs. However, the steady increase of kobs with increasing [Mg2+] up to 0.6 M (Fig. 2, C and D) did not suggest any obvious defects of processing at such high Mg2+ concentrations.

We interpret our results to indicate that (a) the 2'-OH at the RNase P cleavage site is involved in binding of catalytically important Mg2+ as previously suggested (7), and (b) that cleavage chemistry of the modified substrates is inherently slower. Evidence for (a) is the finding that the reductions in rate are most pronounced at lower Mg2+ concentrations. This is seen for the experiments performed at pH 7.0 (Fig. 3A), because at this pH value cleavage efficiencies were high enough to reliably measure rates for the modified ptRNA variants at Mg2+ concentrations in the lower millimolar range.

The relative recovery of catalytic performance (krel) for the modified substrate versions at high [Mg2+] is more pronounced at pH 7.0 than pH 5.5 (for example, compare the krel values at 0.1 M Mg2+ in Fig. 3A versus B and C). We think that two factors contributed to this difference. First, the cleavage rates for the most efficient all-ribose substrate were > 10 min–1 at pH 7.0 and Mg2+ concentrations exceeding 0.04 M and thus at the limit of precise rate determinations by manual kinetics. Therefore, we have to consider some underestimation of krel values at higher Mg2+ concentrations in Fig. 3A. Second, the linear relationship between pH and log kobs for single turnover cleavage of all-ribose ptRNA by M1 RNA starts to level off above ~pH 6.5. Apparently, another step, possibly a conformational change (28), begins to limit the cleavage rate at pH 7.0 for the all-ribose but not for the more slowly cleaved modified substrates. Thus, at pH 7.0 and high Mg2+ concentrations the catalytic defect caused by the 2' modifications at nt –1 seems to be partially masked, whereas results obtained at pH 5.5 provide a better assessment of the catalytic defect, except for ptRNAGlyCNU whose 2'-amino group was inferred to be substantially protonated at pH 5.5 (see below).

Cleavage Site Selection—Next, we analyzed if ptRNAGlyCFU, CNU and CHU were cleaved at the canonical site (–1/+1) by M1 RNA or if the modification directed cleavage to the next unmodified phosphodiester in the 5'-direction (position –2/–1). As shown in Fig. 4, the 2'-F (Fig. 4A, lane 5), 2'-N (Fig. 4B, lane 10) and 2'-H variant (Fig. 4C, lane 14) were cleaved at –1/+1 at pH 7.5. However, the 2'-N variant showed substantial cleavage at position –2/–1 at pH 5.5 (Fig. 4B, lane 12). No substantial cleavage at –2/–1 was detected at pH 5.5 for ptRNAGlyCU, CFU, or CHU (data not shown). Remarkably, the cleavage site in ptRNAGlyCHU was shifted to position –2/–1 when we used the C292 mutant M1 RNA (Fig. 4D, lane 17), which essentially abolishes base pairing with the tRNA CCA terminus (21, 29). In contrast, the all-ribose substrate was still cleaved at position +1/–1 by the C292 mutant RNA (Fig. 4D, lane 18). This documents the synergistic contributions of the 2' substituent at nt –1 and the tRNA 3'-CCA base-pairing interaction to transition state complex formation at the canonical cleavage site, particularly in the context of our substrate that is assumed to form only a weak U+73/U294 interaction with E. coli M1 RNA (30, 31).



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  		FIG. 4.
Cleavage site analysis for ptRNAGly variants carrying single 2'-ribose modifications at nt –1. A, cleavage site analysis for processing of ptRNAGlyCFU by M1 RNA. The 24-mer (lanes 1 and 2) is identical to the first 24 nt of ptRNAGlyCFU (Fig. 1). 24-mer and ptRNAGlyCFU were 32 P-labeled at their 5'-ends. Lane 1, partial nuclease P1 digest of the 24-mer, with the lengths (in nt) of some hydrolysis products marked at the left margin; lanes 2 and 3, partial alkaline hydrolysis (OH–) of the 24-mer (lane 2) or ptRNAGlyCFU (lane 3), with the lengths (in nt) of some hydrolysis products marked at the right margin; the asterisk marks the missing fragment in the alkaline hydrolysis ladders due to the 2'-fluoro modification at nt –1. Note that nuclease P1, as RNase P, generates 5'-phosphate and 3'-OH ends, whereas alkaline hydrolysis produces 5'-OH and 2',3'-cyclic phosphate termini. Lane 5, 5'-cleavage product obtained after processing of ptRNAGlyCFU by M1 RNA at pH 7.5; lane 4, control reaction to that shown in lane 5 (omission of M1 RNA). B, cleavage site analysis for processing of ptRNAGlyCNU by M1 RNA. Lanes 6–8, as lanes 1–3 in panel A (the asterisk on the right side of lane 8 marks the position of the missing 7-nt long alkaline hydrolysis product in lanes 7 and 8); note that nuclease P1 failed to hydrolyze at the site of the 2'-amino modification (marked by the asterisk at the left margin). Lanes 9 and 10, as lanes 4 and 5 in panel A; lanes 11 and 12: as lanes 9 and 10, except that the processing reaction was performed at pH 5.5. On the right margin, only the two 7- and 6-nt long 5'-cleavage product generated by M1 RNA processing are indicated, corresponding to cleavage between –1/+1 and –2/-1, respectively. C, cleavage site analysis for processing of ptRNAGlyCHU by M1 RNA. Lanes 13 and 15, 5'-end-labeled RNA oligonucleotides 5'-pCCCUUU (lane 13) and 5'-pCCCUUUdC (lane 15, the latter carrying a single 2'-deoxy modification at the 3'-terminal C residue) used as length standards, with sizes (in nt) indicated at the left margin; lane 14, 5'-cleavage product obtained after processing of ptRNAGlyCHU by M1 RNA at pH 7.0; lane 16, partial alkaline hydrolysis (OH–) of ptRNAGly CHU, with the sizes of some hydrolysis products marked at the right margin, and the asterisk indicating the missing fragment due to the 2'-deoxy substitution. D, miscleavage (between nt –2/–1) of ptRNAGlyCHU by the G292 to C mutant M1 RNA, but not by wild-type M1 RNA. Lanes 17 and 18, 5'-cleavage product obtained after processing of ptRNAGlyCHU(lane 17) or all-ribose ptRNAGlyCU (lane 18) by the C292 mutant M1 RNA at pH 6.0; lanes 19 and 20, as lanes 15 and 13, respectively, in panel C; lanes 21 and 22, as lanes 17 and 18, but using wild-type M1 RNA; note that 5'-pCCCUUUdC and 5'-pCCCUUUC (both 7-nt long) migrated differently in the denaturing PAA gels (marked by the double arrow at the right margin). Electrophoresis was performed in 30% PAA/8 M urea gels in panels A, B, and C. In panel D, a 25% PAA/8 M urea gel was used and the electrophoresis buffer was 0.5x TBE instead of 1x TBE.

 

pH-dependent Miscleavage of 2'-Amino-modified ptRNA Variants—The pH dependence for processing of ptRNAGlyCNU by M1 RNA revealed substantial miscleavage at pH values < 6.0 (Fig. 5A). The log-linear relationship of the overall cleavage rate and pH with a slope of 1.1 (Fig. 5B) indicates that cleavage chemistry is rate limiting up to pH 8.0 for the ptRNAGlyCNU substrate. As mentioned before, a very similar relationship with the same slope was reported for processing of a ptRNAPhe with a single 2'-H modification at nt –1 by Bacillus subtilis RNase P RNA in the pH range between 6.1 and 7.8 (27). The results in Fig. 5A suggests that protonation of the 2'-amino substituent at low pH causes displacement of catalytic Me2+ due to electrostatic repulsion resulting in utilization of the next unmodified phosphodiester in the 5'-direction. We then analyzed if pH-dependent miscleavage of ptRNAGlyCNU also pertains to a variant (ptRNAGly CNA) with a U to A+73 exchange. This might stabilize the interaction of nt +73 with U294 of E. coli M1 RNA and thus favor cleavage at –1/+1 (30). Fig. 6 shows the similar pH dependence of miscleavage at –2/–1 for ptRNAGlyCNU and ptRNAGlyCNA. Surprisingly, the pH at which 50% miscleavage occurred (termed pH50%), increased by 0.3 pH units from pH 5.3 to 5.6 for ptRNAGlyCNA. Even more surprising, variants with a C to U exchange at nt –1, ptRNAGlyUNA and UNU, showed substantial reductions in miscleavage (Table II). At pH 5.0 and with Mg2+ as the divalent metal ion, these variants were only miscleaved to 53% (UNA) and 14% (UNU), whereas miscleavage amounted to 77% (CNU) and 92% (CNA) for the two other variants. A similar hierarchy of miscleavage was observed in the presence of Mn2+ at pH 5.0 (Table II).



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  		FIG. 6.
pH-dependent miscleavage at nt –2/–1 of ptRNAGlyCNU ({circ}) and ptRNAGlyCNA (•). For experimental details, see legend to Fig. 5A and "Experimental Procedures." The pH at which 50% miscleavage occurred (pH50%) was shifted upwards by 0.3 pH units from pH 5.3 to 5.6 for ptRNAGlyCNA relative to ptRNAGlyCNU. Note that variants ptRNAGlyUNA and ptRNAGlyUNU showed only 53% and 13.7% miscleavage, respectively, at pH 5.0 (see Table II).

 

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  		TABLE II
pH- and metal ion-dependent cleavage site selection in the presence of a 2'-amino substituent at nt -1 and different base identities of nt -1 and +73

The assay conditions are 1 M NH4OAc, 1 or 5 µM E. coli RNase P RNA, <1 nM substrate. Results are based on two to three independent experiments.

 

Direct Metal Ion Coordination to the 2' Substituent at nt –1?—We considered the possibility that the 2' substituent at nt –1 may be involved in direct metal ion coordination, although inner-sphere coordination of 2'-OH ligands to Mg2+ seems to be rare (32).

Unlike Mg2+, transition metal ions, such as Mn2+ and Zn2+, can form stable inner-sphere complexes with nitrogen-containing compounds, for example with the N7 of purine nucleotides (33). A metal ion interaction with the 2' substituent of the G nucleophile was inferred for the group I self-splicing introns from Mn2+ and Zn2+ rescue of a reaction using a 2'-amino-2'-deoxyguanosine analog instead of guanosine as the nucleophile (34). To address the question of a potential direct metal interaction with the 2' substituent at nt –1, we determined the cleavage rate for the 2'-N-modified substrate variants in the presence of Mn2+ versus Mg2+. The results are summarized in Table III and show that cleavage of the all-ribose ptRNA variants by M1 RNA is 2–3-fold less efficient in 10 mM Mn2+ relative to 10 mM Mg2+ (krel = 0.34–0.5). This relation is completely inverted for the substrates with a 2'-N substitution at –1, with ptRNAGlyCNU being processed at an 82-fold higher single turnover rate in the presence of Mn2+ versus Mg2+. This was analyzed at pH 6.5, where cleavage occurred predominantly at –1/+1. The Mn2+ rescue effect was again remarkably influenced by the base identities at –1 and +73, decreasing in the order CNU > CNA > UNU > UNA (Table III). An important reason for these differences is the particularly low cleavage efficiency of the 2'-N-modified variants with a C at –1 in the presence of Mg2+. Could the observed Mn2+ rescue effects be interpreted as indicating a direct metal ion coordination to the 2'-amino substituent at nt –1? We argue that this is not the case because a Mn2+ rescue of similar strength (krel = 52, Table III) was also observed for the ptRNAGlyCHU carrying a 2'-deoxy modification at nt –1, which abrogates a direct metal ion coordination. The absence of a direct metal ion coordination to the 2'-amino substituent at nt –1 is also supported by the inability to stimulate processing of ptRNAGlyCNU by addition of Zn2+ (data not shown).


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  		TABLE III
Mn2+ rescue of ptRNA cleavage in the presence of 2'-ribose modifications at nt -1

The Assay conditions are 1 M NH4OAc, 10 mM Me2+, pH 6.5 (37 °C), 5 µM E. coli RNase P RNA, < 1nM substrate. Results are based on two to three independent experiments.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
2'-Deoxy and 2'-Fluorine Modifications at nt –1—In a previous study by Drew Smith and Norman R. Pace (7), a 2'-deoxy modification at nt –1 was calculated to reduce the rate of catalysis by a factor of 3400, which largely exceeds the factor seen in the present study (~150-fold, Fig. 3B) or in a previous study with the B. subtilis ribozyme (240-fold, Ref. 35). One possible explanation for this discrepancy is that, in the previous study (7), the cleavage rate of an all-ribose ptRNA with a 5'-flank of more than 10 nt and a U residue at nt –1 was compared with the rate for a ptRNA with a single 2'-deoxy A residue as 5' flank. A ptRNA with a 1-nt 5'-flank was estimated to be cleaved up to 30-fold slower than ptRNA variants with 5'-precursor segments of >=2 nt in the reaction catalyzed by B. subtilis RNase P; in addition, changing the nucleotide at position –1 from U to G reduced the cleavage rate 70-fold (36). Likewise, B. subtilis RNase P RNA cleaved a ptRNA with a single 2'-deoxy C as the 5'-flank at a ~30-fold slower rate than a variant thereof with four additional ribonucleotides at the 5'-end (35). Exchanging the nt upstream of the cleavage site from U to A within an 8–10 nt long 5'-flank reduced kcat in the M1 RNA-catalyzed reaction by a factor of 12- to almost 200-fold, depending on the substrate context (13). Even if one assumes that only a minor proportion of these reported reductions in catalytic rate took effect in the previous study (7), such effects on reactivity may easily explain the quantitative differences a 2'-deoxy modification at nt –1 had on the cleavage rate constant in the study by Smith and Pace (3400-fold) relative to the present study (~150-fold, Fig. 3B). It is instructive to compare the results obtained for M1 RNA with those reported for the second large ribozyme model system, the Tetrahymena group I ribozyme. Both RNA enzymes generate cleavage products with 3'-OH termini, in contrast to the known small ribozymes (hammerhead, hairpin, VS, and HDV ribozyme), which produce 2',3'-cyclic phosphates (Ref. 37, and references therein). A difference between the Tetrahymena ribozyme and RNase P RNA, however, is that the former utilizes an exogenous guanosine nucleophile instead of a solvent hydroxide. A 2'-F modification at U-1 of the substrate for the Tetrahymena ribozyme caused a 2.5-fold increase in the Kd for substrate ground state binding and slowed the chemical step by a factor of 8.5 compared with the all-ribose substrate (38). These values are remarkably similar to the effect of a 2'-F modification observed in our study (2.4-fold Kd effect, 10-fold slower chemistry; Table I, Fig. 3B). In the Tetrahymena ribozyme reaction, the cleavage rate was 70-fold faster with a 2'-F versus a 2'-H modification at nt –1 under rate-limiting chemistry. For M1 RNA, this rate advantage of the 2'-F substitution was 13-fold (Fig. 3B), and the 2'-H modification had a slightly more pronounced effect on the Kd of substrate ground state binding in the M1 RNA-catalyzed reaction (7.7-fold versus 3-fold increase; Table I, Ref. 38). Nevertheless, the similarities are striking: (a) in both systems, the 2' modifications at nt –1 had minor to moderate effects on substrate ground state binding, whereas the major effects were on the chemical step; and (b) a 2'-F modification conferred a substantial rate enhancement over the 2'-deoxy substituent, but a 2'-OH function was superior in both systems. Based on this analogy there is no reason not to argue along the same lines as for the Tetrahymena ribozyme (39). The all-ribose substrate reacts 10-fold faster than a derivative with a 2'-F modification at nt –1, because there is donation of a hydrogen bond by the 2'-OH at nt –1 in the transition state (possibly to the 3'-bridging oxygen), although the 2'-OH has a lower electron withdrawing ability. The higher inductive effect of a 2'-F versus 2'-H substituent explains the higher reactivity of the ptRNA with a 2'-F versus 2'-H modification at nt –1. Here we assume that conformational differences of the sugar moieties with different 2' substituents do not play a major role due to the low energy barrier for interconversion (40) between the two major sugar puckering states [C3'-endo (N) and C2'-endo (S)].

Another issue is the hydrogen acceptor function of the 2'-fluoro group, which is predicted based on the availability of lone-pair electrons. There is x-ray structural evidence arguing against fluorine covalently bound to DNA as an effective H bond acceptor (41). This conclusion is based on the finding that a larger proportion of water molecules that form a hydrogen bond to an RNA 2'-OH group are located at 3 Å or less from the 2'-oxygen atom, whereas the majority of water molecules adopt a distance of > 3 Å to a 2'-fluorine despite the smaller van der Waals radius of fluorine relative to oxygen. Also, water molecules around 2'-hydroxyl groups display a more pronounced clustering than those around 2'-fluorines (41). However, hydrogen bonds accepted by a 2'-fluorine from a strong donor, such as a metal ion-coordinated water molecule, are considered to exist with certainty (41). If this kind of hydrogen bonding interaction takes place in the M1 RNA-catalyzed reaction is, however, unknown at present. Thus, it remains a possibility that the 10-fold lower reactivity of ptRNA with a 2'-fluorine relative to a 2'-hydroxyl at nt –1 includes contributions from differences in water structure around the 2' substituent and/or an impaired hydrogen bonding acceptor capacity of the 2'-fluorine. It has been shown for the Tetrahymena ribozyme that a hydrogen bond from the 2'-OH of A207 to the 2'-OH of U-1 stabilizes the transition state (42). Thus, the 2'-OH at nt –1 combines H bonding acceptor and donor functions in catalysis by the Tetrahymena ribozyme. Based on the identical strength of the 2'-fluorine effect (10-fold) in the M1 RNA-catalyzed reaction, it is not unlikely that the 2'-OH at nt –1 not only donates but also accepts a hydrogen bond during catalysis.

Metal Ion Binding at nt –1 and the 2'-Amino Substitution— The defect in Mg2+ binding due to 2' substitutions at nt –1 (Ref. 7; Fig. 3) has led to the formulation of a model where hydrogen bond donation from this 2'-hydroxyl to a metal ion-coordinated water positions the latter for proton donation to the 3'-oxyanion leaving group (7). As outlined below, this model is consistent with our data and represents an alternative to the direct donation of a hydrogen bond from the 2'-OH to the 3'-bridging oxygen in the transtion state. Further evidence for the binding of a metal ion (termed MeB; Refs. 8 and 12) in immediate vicinity of the 2' substituent at nt –1 comes from our results with the 2'-amino modification at this position, where miscleavage of ptRNAGlyCNU(A) at lower pH (Figs. 5 and 6) suggests electrostatic repulsion of metal ion MeB due to protonation of the 2'-amino group. Also, the 2'-amino group would have been expected to result in a much better catalytic performance than the 2'-deoxy modification due to its higher inductive effect and the capability to function in hydrogen bonding. However, ptRNAGlyCNU was cleaved only at a ~2-fold higher rate than ptRNAGlyCHU at pH values >=7.0 (Fig. 3A; data for pH 8.0 not shown) where protonation of the 2'-amino group is expected to be negligible. Several reasons may contribute to the low catalytic performance of ptRNAGlyCNU. For example, the 2'-N substituent may constrain the conformation of the ribose at nt –1, may create an artificial interaction or may have unfavourable effects on water structure around this functional group. It is interesting to note in this context that the 2'-amino modification also blocked hydrolysis by RNase A, although this reaction follows a different mechanism (nucleophilic attack of the 2' substituent on phosphorus) than the one catalyzed by RNase P. It was proposed that steric factors, possibly related to the favored C2'-endo pucker of the nucleoside analog, may prevent the 2'-amino group from being favorably placed in the RNase A active site for nucleophilic attack on phosphorus (43, 44).

Remarkable is our finding (Table II) that a C to U exchange at nt –1 reduced miscleavage at pH 5.0 from 77% (ptRNAGlyCNU) to 14% (ptRNAGlyUNU) or from 92% (ptRNAGlyCNA) to 53% (ptRNAGlyUNA). This correlates with the very low single turnover rate of cleavage for substrates with a 2'-amino-2'-deoxy C residue at –1 and in the presence of Mg2+ (Table III). One explanation could be that effective electrostatic repulsion of MgB (Fig. 7) requires simultaneous protonation of the 2'-amino group at nt –1 and of the cytidine N3 function (unperturbed pKa: ~4.5; Ref. 40). The pKa of the N3 function may be significantly perturbed, as observed by NMR for a cytidine in a ribosomal A loop module (apparent pKa ~6.4), where N3 protonation also caused subtle conformational changes of A loop structure (45). Likewise, protonation of adenosine N1 (unperturbed pKa close to 4.0; Ref. 40) may be the reason for increased miscleavage when U+73 is exchanged for A (Table II). In summary, our results document an unexpectedly strong dependence of miscleavage on the base identities at nt –1 and +73, with miscleavage favored in the order C-1 > U-1 and A73 > U73, suggesting that the 2' substituent at nt –1, catalytic metal ion cofactors as well as base functional groups of nt –1 and +73 constitute an intricate and sensitive network in the active site, consistent with results from a recent in depth analysis of the interaction between the substrate nt +73 and nt 294 of M1 RNA (31).



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  		FIG. 7.
Active site and transition state model of E. coli M1 RNA-substrate complexes. Bases –1, +73, and +75 of the ptRNA substrate as well as G292 of M1 RNA are depicted in green; green arrows illustrate that base identities at –1 and +73 as well as disruption of the base-pairing between C+75 and G292 had profound effects on the kinetic phenotype, metal ion utilization, and cleavage site selection (Fig. 4, Tables II and III) of substrates with single 2' modifications at nt –1 (represented by the 2'-OH of the all-ribose substrate shown in blue). Putative, catalytically important Mg2+ ions are shown in magenta. For the Mg2+ close to G292/G293/U294 in the L15/16 loop of M1 RNA, see Ref. 12, and references therein. The Watson-Crick base pairing interaction between G292/G293 of M1 RNA and C+74/+C75 of tRNA (21, 29) as well as the proposed interaction between U294 of M1 RNA and U+73 of tRNA (30) have been depicted. Nt A248, A249, C252, C253, G332, and A333 were shown to be in close vicinity of the substrate nt –1 in E·S complexes, as revealed by short range cross-links (49, 50). It should be mentioned here that Michael Harris and co-workers recently reported evidence (51) that the conserved A248 specifically interacts with the nucleotide at –1 (indicated by the dashed line). The transition state model for hydrolysis of the scissile phosphodiester connecting nt +1 and –1 is derived from that proposed in (52), according to which MgB directly coordinates to the pro-Rp phosphate oxygen and simultaneously interacts with the OH– nucleophile (in orange) and the 2'-OH at position –1. Alternatively, both MgA and MgB may directly coordinate to the pro-Rp oxygen, but MgA instead of MgB coordinates the OH– nucleophile (8, 53), as indicated by the question mark next to MgA. Sulfur substitutions at the canonical cleavage site, which essentially abolished catalysis in the presence of Mg2+, are depicted (S with crossed out red circle). M1 RNA-catalyzed cleavage of a substrate with a single sulfur substitution of the pro-Rp oxygen could be rescued by replacing Mg2+ with Mn2+ or Cd2+, whereas no such rescue could be obtained for a sulfur substitution of the pro-Sp oxygen (marked Sp); the Sp-phosphorothioate modification also weakened substrate ground state binding 30-fold (8). Sulfur substitution of the 3'-bridging oxygen abolished cleavage in the presence of Mg2+ as well as thiophilic metal ions, such as Mn2+ or Cd2+ (54). The aforementioned findings do not rule out metal ion interactions with the pro-Sp oxygen and the 3'-bridging oxygen, as denoted by question marks.

 

In any case, a 2'-amino modification at nt –1 showed fundamentally different effects in reactions catalyzed by E. coli M1 RNA (this study) and the Tetrahymena ribozyme (39), thus revealing substantial differences in transition state complex geometry for the two ribozymes. In the Tetrahymena ribozyme reaction, a substrate with a 2'-amino modification at nt –1 (termed rSN) reacted even faster than the all-ribose substrate (rSOH) at lower pH (between pH 4.5 and 6.5), i.e. under conditions where a substantial proportion of the 2'-amino group is protonated (the apparent pKa was determined to be 5.0; Ref. 39). These findings ruled out that the 2' substituent at nt –1 is involved in binding of a metal ion, because a NH3+ group has no lone pair electrons for a direct metal ion interaction and the positive charge would have been expected to repulse a metal ion. Furthermore, the rSN substrate was cleaved with roughly the same catalytic efficiency as rSOH at pH 7.0, where protonation is low or negligible and another step than the chemical starts to become rate-limiting for cleavage of rSOH (39). In contrast to the Tetrahymena ribozyme, protonation of the 2'-amino group in ptRNAGlyCNU was apparently detrimental to cleavage in the reaction catalyzed by M1 RNA. Also and again in contrast to the Tetrahymena ribozyme, ptRNAGlyCNU was cleaved at extremely low rates at pH 7.0, where, analogous to the Tetrahymena ribozyme reaction (see above), another step than the chemical begins to become rate-limiting for cleavage of the all-ribose substrate. In conclusion, all existing experimental evidence supports the notion that a metal ion binds next to the 2' substituent at nt –1 in the reaction catalyzed by bacterial RNase P RNA, but not in that catalyzed by the Tetrahymena ribozyme.

RNase P RNA Active Site Plasticity—Our results have demonstrated a close and sensitive communication between the 2' substituent at nt –1, the nature (Mg2+ or Mn2+) of catalytic divalent metal ion(s) and, surprisingly, the base functional groups of nt –1 and +73 (Tables II and III; Figs. 6 and 7), suggesting a rather dynamic structure of catalytic M1 RNA-substrate complexes. The disruption of the C+75/G292 base pair in E·S complexes caused miscleavage of ptRNAGlyCHU but not of all-ribose ptRNAGlyCU (Fig. 4D), demonstrating the communication between the 2' substituent at nt –1 and the tRNA 3'-end "NCCA" interaction with M1 RNA. Our results are even more remarkable since our substrate, a bacterial ptRNAGly, is a simple class I tRNA with a canonical 7-bp acceptor stem. In comparison, another natural ptRNA substrate that has been extensively characterized, namely E. coli ptRNATyrSu3 (2), is more complex because it has the capacity to form a 9-bp acceptor stem and its specific 5'-precursor sequence plays an important role in complex formation with M1 RNA (46). Irrespective of such differences in substrate nature, our findings are consistent with those of Leif Kirsebom and co-workers (reviewed in Refs. 2, 13, 31, and references therein) who have characterized in large detail the intricate and flexible interplay of metal ion cofactors with several structural determinants in the substrate and ribozyme near the cleavage site. The emerging picture of a highly dynamic nature of the RNase P RNA catalytic site correlates with the peculiar biological role of the enzyme. Bacterial RNase P (RNA) differs from other ribozymes, such as the self-splicing group I introns, in its natural trans-cleavage activity and its biologically important capacity to recognize a large variety of ptRNAs, even including non-tRNA substrates in some organisms (reviewed in Ref. 3). The enzyme has to act with similar efficiency on ptRNAs carrying unrelated 5'-flanking sequences and which differ in structural details of their mature domains. Some ptRNAs even have to be processed at a site shifted by 1 nt in order to generate a mature tRNA product with an 8-bp instead of a 7-bp long acceptor stem (tRNAHis, tRNASeCys; Refs. 47 and 48). This broad substrate specificity is thought to have brought about the consequence of a rather flexible active site architecture compared with other large but more specialized ribozymes, such as the group I self-splicing introns. The dynamic nature of RNase P RNA is also a likely explanation for the difficulties associated with attempts to crystallize this ribozyme, to define the constituents of its active site architecture and to pinpoint the number and exact location of catalytic metal ion cofactors.


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Grant HA 1672/7-3. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Current address: Lund University, Dept. of Chemistry, Organic Chemistry 1, P.O. Box 124, SE-221 00, Lund, Sweden. Back

Current address: Philipps-Universität Marburg, Institut für Pharmazeutische Chemie, Marbacher Weg 6, D-35037, Marburg, Germany. Back

|| To whom correspondence should be addressed. Tel.: 6421-28-25827; Fax: 6421-28-25854; E-mail: roland.hartmann{at}staff.uni-marburg.de.

1 The abbreviations used are: RNase P, ribonuclease P; nt, nucleotide; MES, 4-morpholineethanesulfonic acid; PIPES, 1,4-piperazinedi-ethanesulfonic acid; HEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; TBE, Tris borate/EDTA. Back


   ACKNOWLEDGMENTS
 
We thank Rita Held and Sybille Siedler for excellent technical assistance, and Silke Busch for contributing to the experiments shown in Fig. 4. We are grateful to Leif A. Kirsebom for the numerous fruitful discussions and exchange of results before publication.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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