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Role of metal ions in the hydrolysis reaction catalyzed by RNase P RNA from Bacillus subtilis1

https://doi.org/10.1006/jmbi.1999.2890Get rights and content

Abstract

Precursor tRNA (ptRNA) substrates carrying a single Rp or Sp-phosphorothioate modification at the RNase P cleavage site were used as tools to study the cleavage mechanism of RNase P RNA from Bacillus subtilis. Both the Sp and the Rp-diastereomer reduced the rate of processing at least 104-fold under conditions where the chemical step is essentially rate-limiting. Neither the Rp nor the Sp-phosphorothioate modification affected ptRNA ground state binding to B. subtilis RNase P RNA. Processing of the Rp-diastereomeric ptRNA could be restored in the presence of Mn2+ or Cd2+, demonstrating direct metal ion coordination to the pro-Rp oxygen during catalysis. With Cd2+, processing required the presence of another metal ion, such as Ca2+ or Mg2+, to mediate substrate binding. This is in contrast to Escherichia coli RNase P RNA, which promotes cleavage of Rp-diastereomeric ptRNA in the presence of Cd2+ as the sole divalent metal ion. Analysis of [Cd2+]-dependent processing of the Rp-diastereomeric substrate by B. subtilis RNase P RNA was consistent with the involvement of at least two metal ions in catalysis. The presence of two catalytic metal ion binding sites is also supported by the inhibition mode of Ca2+ on cleavage of unmodified ptRNA. In the presence of an Sp-phosphorothioate modification at the scissile bond, neither Mn2+ nor Cd2+ were able to restore significant cleavage at this location. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Unaffected ground state binding of the Sp-diastereomeric ptRNA but a ⩾104-fold reduced hydrolysis rate may indicate a crucial role of thepro-Sp oxygen in transition state stabilization or may be attributed to steric exclusion of catalytic metal ions. Based on our comparative analyses of B. subtilis and E. coli RNase P RNA, each representing the main structural subtypes of bacterial RNase P RNA, common features in terms of active site constraints and role of catalytic metal ions can now be formulated for bacterial RNase P RNAs. On the other hand, substantial and unexpected differences with respect to the overall metal ion requirements and tRNA binding modes have been observed for the two catalytic RNAs.

Introduction

Ribonuclease P (RNase P) is an essential structure-specific endoribonuclease that generates the mature 5′ ends of tRNAs. In vitro, RNA subunits of bacterial RNase P enzymes were shown to be catalytically active in the absence of the protein subunit (Guerrier-Takada et al., 1983). Processing of precursor tRNAs (ptRNAs) by RNase P is an essentially irreversible reaction yielding 3′-OH and 5′-phosphate termini. A hydroxide ion coordinated to a metal ion and/or metal ion hydrate (Smith & Pace, 1993), or a water molecule activated by a metal-bound hydroxide (Guerrier-Takada et al., 1986), is thought to act as the nucleophile in an SN2 in-line displacement mechanism.

We have recently studied the cleavage mechanism of RNase P RNA from Escherichia coli, using ptRNA substrates carrying a single Rp or Sp-phosphorothioate modification at the RNase P cleavage site (Warnecke et al., 1996). Both the Sp and the Rp-diastereomer reduced the rate of processing by E. coli RNase P RNA at least 1000-fold under conditions where the chemical step is rate-limiting. The Rp-modification had no and the Sp-modification a moderate effect (30-fold reduced affinity) on ptRNA ground state binding to RNase P RNA under the conditions tested. Processing of the Rp-diastereomeric substrate was largely restored in the presence of the “thiophilic” Cd2+ as the only divalent metal ion, demonstrating direct metal ion coordination to the (pro)-Rp substituent at the cleavage site. The cooperative dependence upon [Cd2+] (Hill coefficient nH = 1.8) is consistent with a two-metal-ion mechanism. Our results did not provide evidence for a specific role of Mg2+ at the pro-Sp oxygen. In the presence of the Sp-modification, neither Mn2+ nor Cd2+ were able to restore detectable cleavage at the canonical site. Instead, the ribozyme promoted cleavage at the neighboring unmodified phosphodiester with low efficiency. Direct metal ion coordination to the (pro)-Rp substituent during catalysis by E. coli RNase P RNA was also inferred from Mn2+ rescue experiments using another ptRNA substrate with an Rp-phosphorothioate modification at the cleavage site (Chen et al., 1997).

RNase P RNA from Bacillus subtilis (abbreviated as P RNA in the following) has been the second important model system to study bacterial RNase P RNA structure and function (e.g. see Guerrier-Takada et al 1983, Gardiner et al 1985, Reich et al 1988, Smith et al 1992, Beebe and Fierke 1994, Kurz et al 1998, LaGrandeur et al 1994, Pan and Zhong 1994, Pan 1995, Pan et al 1995, Beebe et al 1996, Loria and Pan 1996, Loria and Pan 1997, Loria and Pan 1998, SZarrinkar et al 1996, Crary et al 1998), which has gained further importance since the X-ray structure of the B. subtilis RNase P protein component has become available (Stams et al., 1998). Large differences exist on the secondary structural level between “type A” E. coli-like and “type B” B. subtilis-like RNase P RNAs Brown and Pace 1992, Haas et al 1996. In addition, B. subtilis P RNA exhibits salt requirements distinct from those of E. coli RNase P RNA Guerrier-Takada et al 1983, Guerrier-Takada et al 1986, Reich et al 1988, and it is not clear yet how similar the kinetics and thermodynamics of ptRNAs cleavage catalyzed by the two ribozymes are (e.g. see Tallsjo and Kirsebom 1993, Beebe and Fierke 1994). Here we have investigated in more detail the role of metal ions in catalysis by B. subtilis P RNA, which we consider as a prerequisite in order to define mechanistic similarities and differences between the two RNase P ribozyme subclasses, to identify idiosyncrasies of each system, and to be able to draw more general conclusions about the role of metal ions in reactions catalyzed by bacterial RNase P ribozymes. At least one metal ion has been proposed to be involved in catalysis by B. subtilis P RNA (Beebe et al., 1996). As in the previous study on the E. coli ribozyme (Warnecke et al., 1996), we used ptRNA substrates carrying a single Rp or Sp-phosphorothioate modification at the RNase P cleavage site as tools to study the cleavage mechanism of B. subtilis P RNA.

Section snippets

ptRNA binding to B. subtilis P RNA

Precursor tRNAGly substrates carried a 7 nt 5′ flank and either an unmodified phosphodiester, or a single Rp or Sp-phosphorothioate modification (Figure 1) at the RNase P cleavage site. We first analyzed if the phosphorothioate modifications had an effect on specific binding of ptRNAGly to B. subtilis P RNA, using a gel retardation method Hardt et al 1993, Hardt et al 1995 as well as a gel filtration centrifuge column assay Beebe and Fierke 1994, Beebe et al 1996. Binding experiments

Direct metal ion coordination to the pro-Rp oxygen

The strong rescue of cleavage of the Rp-diastereomeric ptRNA in the presence of Cd2+, indicating direct metal ion coordination to the pro-Rp oxygen at the scissile phosphodiester, has now been observed in reactions catalyzed by ribozymes from B. subtilis (this study) and E. coli (Warnecke et al., 1996) as well as by the E. coli holoenzyme (Warnecke et al., 1997). With both ribozymes, a bulky sulfur substitution at this position had no measurable effect on RNase P RNA-ptRNA complex formation (

Conclusions

This study addressed the role of catalytic metal ions in the B. subtilis P RNA cleavage reaction, the second prominent bacterial RNase P model system. The architecture of B. subtilis P RNA largely differs from “ E. coli-like” RNase P RNAs. Both types of RNase P RNA apparently utilize different peripheral structural elements to stabilize a similar catalytic core structure Chen et al 1998, Massire et al 1998. With respect to the cleavage mechanism, the following results were in line with those

DNA templates and preparation of RNAs

RNAs were obtained by in vitro run-off transcription using T7 RNA polymerase as described Schlegl et al 1992, Hardt et al 1993, Hardt et al 1996. P RNA from B. subtilis was transcribed from plasmid pDW66 (Smith et al., 1992)linearized with DraI. The 3′ portion (starting at G+18) of Thermus thermophilus tRNAGly was prepared by T7 transcription in the presence of 10 mM GMP/2.5 mM GTP essentially as described (Hardt et al., 1993)using a PCR template amplified from plasmid pTT675 (Vogel et al.,

Acknowledgements

We are grateful to Andrea Eickmann, Jens Peter Fürste, Rolf Bald and Volker A. Erdmann for the synthesis and purification of RNA oligonucleotides with single phosphorothioate modifications, and James W. Brown and Norman R. Pace for providing plasmid pDW66. Financial support for these studies from the Deutsche Forschungsgemeinschaft (Ha 1672/7–1/7–2) is acknowledged.

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    Edited by A. R. Fersht

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    Present address: J. M. Warnecke, Universität Witten/Herdecke, Institut für Molekularbiologie, Stockumer Str. 10, D-58453 Witten, Germany.

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