Minor changes largely restore catalytic activity of archaeal RNase P RNA from Methanothermobacter thermoautotrophicus

The increased protein proportion of archaeal and eukaryal ribonuclease (RNase) P holoenzymes parallels a vast decrease in the catalytic activity of their RNA subunits (P RNAs) alone. We show that a few mutations toward the bacterial P RNA consensus substantially activate the catalytic (C-) domain of archaeal P RNA from Methanothermobacter, in the absence and presence of the bacterial RNase P protein. Large increases in ribozyme activity required the cooperative effect of at least two structural alterations. The P1 helix of P RNA from Methanothermobacter was found to be extended, which increases ribozyme activity (ca 200-fold) and stabilizes the tertiary structure. Activity increases of mutated archaeal C-domain variants were more pronounced in the context of chimeric P RNAs carrying the bacterial specificity (S-) domain of Escherichia coli instead of the archaeal S-domain. This could be explained by the loss of the archaeal S-domain''s capacity to support tight and productive substrate binding in the absence of protein cofactors. Our results demonstrate that the catalytic capacity of archaeal P RNAs is close to that of their bacterial counterparts, but is masked by minor changes in the C-domain and, particularly, by poor function of the archaeal S-domain in the absence of archaeal protein cofactors.     

Modular construction for function of a ribonucleoprotein enzyme: the catalytic domain of Bacillus subtilis RNase P complexed with B. subtilis RNase P protein

The bacterial RNase P holoenzyme catalyzes the formation of the mature 5′-end of tRNAs and is composed of an RNA and a protein subunit. Among the two folding domains of the RNase P RNA, the catalytic domain (C-domain) contains the active site of this ribozyme. We investigated specific binding of the Bacillus subtilis C-domain with the B.subtilis RNase P protein and examined the catalytic activity of this C-domain–P protein complex. The C-domain forms a specific complex with the P protein with a binding constant of ~0.1 µM. The C-domain–P protein complex and the holoenzyme are equally efficient in cleaving single-stranded RNA (~0.9 min^–1 at pH 7.8) and substrates with a hairpin–loop 3′ to the cleavage site (~40 min^–1). The holoenzyme reaction is much more efficient with a pre-tRNA substrate, binding at least 100-fold better and cleaving 10–500 times more efficiently. These results demonstrate that the RNase P holoenzyme is functionally constructed in three parts. The catalytic domain alone contains the active site, but has little specificity and affinity for most substrates. The specificity and affinity for the substrate is generated by either the specificity domain of RNase P RNA binding to a T stem–loop-like hairpin or RNase P protein binding to a single-stranded RNA. This modular construction may be exploited to obtain RNase P-based ribonucleoprotein complexes with altered substrate specificity.     

Effective Inhibition of Human Immunodeficiency Virus 1 Replication by Engineered RNase P Ribozyme

Using an in vitro selection procedure, we have previously isolated RNase P ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, a variant was used to target the HIV RNA sequence in the tat region. The variant cleaved the tat RNA sequence in vitro about 20 times more efficiently than the wild type ribozyme. Our results provide the first direct evidence that combined mutations at nucleotide 83 and 340 of RNase P catalytic RNA from Escherichia coli (G₈₃ -> U₈₃ and G₃₄₀ -> A₃₄₀) increase the overall efficiency of the ribozyme in cleaving an HIV RNA sequence. Moreover, the variant is more effective in reducing HIV-1 p24 expression and intracellular viral RNA level in cells than the wild type ribozyme. A reduction of about 90% in viral RNA level and a reduction of 150 fold in viral growth were observed in cells that expressed the variant, while a reduction of less than 10% was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, engineered ribozyme variants are effective in inhibiting HIV infection. These results also demonstrate the potential of engineering RNase P ribozymes for anti-HIV application.     

Differential effects of the protein cofactor on the interactions between an RNase P ribozyme and its target mRNA substrate

RNase P from Escherichia coli is a tRNA-processing enzyme and consists of a catalytic RNA subunit (M1 RNA) and a protein component (C5 protein). M1GS, a gene-targeting ribozyme derived from M1, can cleave a herpes simplex virus 1 mRNA efficiently in vitro and inhibit its expression effectively in viral-infected cells. In this study, the effects of C5 on the interactions between a M1GS ribozyme and a model mRNA substrate were investigated by site-specific UV crosslink mapping. In the presence of the protein cofactor, the ribozyme regions crosslinked to the substrate sequence 3′ immediately to the cleavage site were similar to those found in the absence of C5. Meanwhile, some of the ribozyme regions (e.g. P12 and J11/12) that were crosslinked to the leader sequence 5′ immediately to the cleavage site in the presence of C5 were different from those regions (e.g. P3 and P4) found in the absence of the protein cofactor and were not among those that are believed to interact with a tRNA. Understanding how C5 affects the specific interactions between the ribozyme and its target mRNA may facilitate the development of gene-targeting ribozymes that function effectively in vivo, in the presence of cellular proteins.     

Core requirements for glmS ribozyme self-cleavage reveal a putative pseudoknot structure

The glmS ribozyme is a self-cleaving RNA catalyst that resides in the 5′-untranslated region of glmS mRNA in certain bacteria. The ribozyme is specifically activated by glucosamine-6-phosphate (GlcN6P), the metabolic product of the GlmS protein, and is thus proposed to provide a feedback mechanism of riboswitch regulation. Both phylogenetic and biochemical analyses of the glmS ribozyme have established a highly conserved core sequence and secondary structure required for GlcN6P-dependent self-cleavage. However, the high degree of nucleotide conservation offers few clues regarding the higher-order structural organization of the catalytic core. To further investigate core ribozyme structure, minimal ‘consensus-type’ glmS ribozymes that retain GlcN6P-dependent activity were produced. Mutational analyses of consensus-type glmS ribozymes support a model for core ribozyme folding through a pseudoknot structure formed by the interaction of two highly conserved sequence segments. Moreover, GlcN6P-dependent function is demonstrated for bimolecular constructs in which substrate interaction with the ribozyme is minimally comprised of sequence representing that involved in putative pseudoknot formation. These studies suggest that the glmS ribozyme adopts an intricate multi-strand catalytic core through the formation of a pseudoknot structure, and provide a refined model for further considering GlcN6P interaction and GlcN6P-dependent ribozyme function.     

Chloroplast ribonuclease P does not utilize the ribozyme-type pre-tRNA cleavage mechanism

The transfer RNA 5' maturation enzyme RNase P has been characterized in Bacteria, Archaea, and Eukarya. The purified enzyme from all three kingdoms is a ribonucleoprotein containing an essential RNA subunit; indeed, the RNA subunit of bacterial RNase P RNA is the sole catalytic component. In contrast, the RNase P activity isolated from spinach chloroplasts lacks an RNA component and appears to function as a catalytic protein. Nonetheless, the chloroplast enzyme recognizes a pre-tRNA substrate for E. coli RNase P and cleaves it as efficiently and precisely as does the bacterial enzyme. To ascertain whether there are differences in catalytic mechanism between an all-RNA and an all-protein RNase P, we took advantage of the fact that phosphodiester bond selection and hydrolysis by the E. coli RNase P ribozyme is directed by a Mg2+ ion coordinated to the nonbridging pro-Rp oxygen of the scissile bond, and is blocked by sulfur replacement of this oxygen. We therefore tested the ability of the chloroplast enzyme to process a precursor tRNA containing this sulfur substitution. Partially purified RNase P from spinach chloroplasts can accurately and efficiently process phosphorothioate-substituted pre-tRNAs; cleavage occurs exclusively at the thio-containing scissile bond. The enzymatic throughput is fivefold slower, consistent with a general chemical effect of the phosphorothioate substitution rather than with a metal coordination deficiency. The chloroplast RNase P reaction mechanism therefore does not involve a catalytic Mg2+ bonded to the pro-Rp phosphate oxygen, and hence is distinct from the mechanism of the bacterial ribozyme RNase P.     

Mapping metal-binding sites in the catalytic domain of bacterial RNase P RNA

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that contains a universally conserved, catalytically active RNA component. RNase P RNA requires divalent metal ions for folding, substrate binding, and catalysis. Despite recent advances in understanding the structure of RNase P RNA, no comprehensive analysis of metal-binding sites has been reported, in part due to the poor crystallization properties of this large RNA. We have developed an abbreviated yet still catalytic construct, Bst P7Δ RNA, which contains the catalytic domain of the bacterial RNase P RNA and has improved crystallization properties. We use this mutant RNA as well as the native RNA to map metal-binding sites in the catalytic core of the bacterial RNase P RNA, by anomalous scattering in diffraction analysis. The results provide insight into the interplay between RNA structure and focalization of metal ions, and a structural basis for some previous biochemical observations with RNase P. We use electrostatic calculations to extract the potential functional significance of these metal-binding sites with respect to binding Mg²⁺. The results suggest that with at least one important exception of specific binding, these sites mainly map areas of diffuse association of magnesium ions.     

UV cross-link mapping of the substrate-binding site of an RNase P ribozyme to a target mRNA sequence.

RNase P ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural ptRNA substrate. When covalently linked with a guide sequence, the ribozyme can function as a sequence-specific endonuclease and cleave any target RNA sequences that base pair with the guide sequence. Using a site-directed ultraviolet (UV) cross-linking approach, we have mapped the regions of the ribozyme that are in close proximity to a substrate that contains the mRNA sequence encoding thymidine kinase of human herpes simplex virus 1. Our data suggest that the cleavage site of the mRNA substrate is positioned at the same regions of the ribozyme that bind to the cleavage site of a ptRNA. The mRNA-binding domains include regions that interact with the acceptor stem and T-stem and in addition, regions that are unique and not in close contact with a ptRNA. Identification of the mRNA-binding site provides a foundation to study how RNase P ribozymes achieve their sequence specificity and facilitates the development of gene-targeting ribozymes.     

RNase P ribozyme inhibits cytomegalovirus replication by blocking the expression of viral capsid proteins

By linking a guide sequence to the catalytic RNA subunit of RNase P (M1 RNA), we constructed a functional ribozyme (M1GS RNA) that targets the overlapping mRNA region of two human cytomegalovirus (HCMV) capsid proteins, the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. The ribozyme efficiently cleaved the target mRNA sequence in vitro. Moreover, a reduction of >85% in the expression of CSP and assemblin and a reduction of 4000-fold in viral growth were observed in the HCMV-infected cells that expressed the functional ribozyme. In contrast, there was no significant reduction in viral gene expression and growth in virus-infected cells that either did not express the ribozyme or produced a ‘disabled’ ribozyme carrying mutations that abolished its catalytic activity. Characterization of the effects of the ribozyme on the HCMV lytic replication cycle further indicates that the expression of the functional ribozyme specifically inhibits the expression of CSP and assemblin, and consequently blocks viral capsid formation and growth. Our results provide the direct evidence that RNase P ribozymes can be used as an effective gene-targeting agent for antiviral applications, including abolishing HCMV growth by blocking the expression of the virus-encoded capsid proteins.     

Tailoring the switch from IRES-dependent to 5′-end-dependent translation with the RNase P ribozyme

Translation initiation driven by internal ribosome entry site (IRES) elements is dependent on the structural organization of the IRES region. We have previously shown that a structural motif within the foot-and-mouth-disease virus IRES is recognized in vitro as substrate for the Synechocystis sp. RNase P ribozyme. Here we show that this structure-dependent endonuclease recognizes the IRES element in cultured cells, leading to inhibition of translation. Inhibition of IRES activity was dependent on the expression of the active ribozyme RNA subunit. Moreover, expression of the antisense sequence of the ribozyme did not inhibit IRES activity, demonstrating that stable RNA structures located upstream of the IRES element do not interfere with internal initiation. RNAs carrying defective IRES mutants that were substrates of the ribozyme in vivo revealed an increased translation of the reporter in response to the expression of the active ribozyme. In support of RNA cleavage, subsequent analysis of the translation initiation manner indicated a switch from IRES-dependent to 5′-end-dependent translation of RNase P target RNAs. We conclude that the IRES element is inactivated by expression in cis of RNase P in the cytoplasm of cultured cells, providing a promising antiviral tool to combat picornavirus infections. Furthermore, our results reinforce the essential role of the structural motif that serves as RNase P recognition motif for IRES activity.     

Phylogenetic-comparative analysis of the eukaryal ribonuclease P RNA

Ribonuclease P (RNase P) is the ribonucleoprotein enzyme that cleaves 5'-leader sequences from precursor-tRNAs. Bacterial and eukaryal RNase P RNAs differ fundamentally in that the former, but not the latter, are capable of catalyzing pre-tRNA maturation in vitro in the absence of proteins. An explanation of these functional differences will be assisted by a detailed comparison of bacterial and eukaryal RNase P RNA structures. However, the structures of eukaryal RNase P RNAs remain poorly characterized, compared to their bacterial and archaeal homologs. Hence, we have taken a phylogenetic-comparative approach to refine the secondary structures of eukaryal RNase P RNAs. To this end, 20 new RNase P RNA sequences have been determined from species of ascomycetous fungi representative of the genera Arxiozyma, Clavispora, Kluyveromyces, Pichia, Saccharomyces, Saccharomycopsis, Torulaspora, Wickerhamia, and Zygosaccharomyces. Phylogenetic-comparative analysis of these and other sequences refines previous eukaryal RNase P RNA secondary structure models. Patterns of sequence conservation and length variation refine the minimum-consensus model of the core eukaryal RNA structure. In comparison to bacterial RNase P RNAs, the eukaryal homologs lack RNA structural elements thought to be critical for both substrate binding and catalysis. Nonetheless, the eukaryal RNA retains the main features of the catalytic core of the bacterial RNase P. This indicates that the eukaryal RNA remains intrinsically a ribozyme.     

Substrate shape specificity of E coli RNase P ribozyme is dependent on the concentration of magnesium ion

The bacterial RNase P ribozyme can accept a hairpin RNA with CCA-3' tag sequence as well as a cloverleaf pre-tRNA as substrate in vitro, but the details are not known. By switching tRNA structure using an antisense guide DNA technique, we examined the Escherichia coli RNase P ribozyme specificity for substrate RNA of a given shape. Analysis of the RNase P reaction with various concentrations of magnesium ion revealed that the ribozyme cleaved only the cloverleaf RNA at below 10 mM magnesium ion. At 10 mM magnesium ion or more, the ribozyme also cleaved a hairpin RNA with a CCA-3' tag sequence. At above 20 mM magnesium ion, cleavage site wobbling by the enzyme in tRNA-derived hairpin occurred, and the substrate specificity of the enzyme became broader. Additional studies using another hairpin substrate demonstrated the same tendency. Our data strongly suggest that raising the concentration of metal ion induces a conformational change in the RNA enzyme.     

Analysis of the tertiary structure of the ribonuclease P ribozyme-substrate complex by site-specific photoaffinity crosslinking.

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well-established, and a low-resolution model of the three-dimensional structure of the ribozyme-substrate complex has been proposed based on site-specific crosslinking and phylogenetic comparative data [Harris ME et al., 1994 EMBO J 13:3953-3963]. However, several substructures of that model were poorly constrained by the available data. In the present analysis, additional constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. Circularly permuted RNase P RNAs were used to position an azidophenacyl photoactive crosslinking agent specifically at strategic sites within the ribozyme-substrate complex. Crosslink sites were mapped by primer extension and confirmed by analysis of the mobility of the crosslinked RNA lariats on denaturing acrylamide gels relative to circular and linear RNA standards. Crosslinked species generally retained significant catalytic activity, indicating that the results reflect the native ribozyme structure. The crosslinking results support the general configuration of the structure model and predicate new positions and orientations for helices that were previously poorly constrained by the data set. The expanded library of crosslinking constraints was used, together with secondary and tertiary structure identified by phylogenetic sequence comparisons, to refine significantly the model of RNase P RNA with bound substrate pre-tRNA. The crosslinking results and data from chemical-modification and mutational studies are discussed in the context of the current structural perspective on this ribozyme.     

Molecular recognition properties of IGS-mediated reactions catalyzed by a Pneumocystis carinii group I intron

We report the development, analysis and use of a new combinatorial approach to analyze the substrate sequence dependence of the suicide inhibition, cyclization, and reverse cyclization reactions catalyzed by a group I intron from the opportunistic pathogen Pneumocystis carinii. We demonstrate that the sequence specificity of these Internal Guide Sequence (IGS)-mediated reactions is not high. In addition, the sequence specificity of suicide inhibition decreases with increasing MgCl₂ concentration, reverse cyclization is substantially more sequence specific than suicide inhibition, and multiple reverse cyclization products occur, in part due to the formation of multiple cyclization intermediates. Thermodynamic analysis reveals that a base pair at position –4 of the resultant 5′ exon–IGS (P1) helix is crucial for tertiary docking of the P1 helix into the catalytic core of the ribozyme in the suicide inhibition reaction. In contrast to results reported with a Tetrahymena ribozyme, altering the sequence of the IGS of the P.carinii ribozyme can result in a marked reduction in tertiary stability of docking the resultant P1 helix into the catalytic core of the ribozyme. Finally, results indicate that RNA targeting strategies which exploit tertiary interactions could have low specificity due to the tolerance of mismatched base pairs.     

Structural modeling of RNase P RNA of the hyperthermophilic archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is a ubiquitous trans-acting ribozyme that processes the 5′ leader sequence of precursor tRNA (pre-tRNA). The RNase P RNA (PhopRNA) of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 is central to the catalytic process and binds five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) which contribute to the enzymatic activity of the holoenzyme. Despite significant progress in determining the crystal structure of the proteins, the structure of PhopRNA remains elusive. Comparative analysis of the RNase P RNA sequences and existing crystallographic structural information of the bacterial RNase P RNAs were combined to generate a phylogenetically supported three-dimensional (3-D) model of the PhopRNA. The model structure shows an essentially flat disk with 16 tightly packed helices and a conserved face suitable for the binding of pre-tRNA. Moreover, the structure in solution was investigated by enzymatic probing and small-angle X-ray scattering (SAXS) analysis. The low resolution model derived from SAXS and the comparative 3-D model have similar overall shapes. The 3-D model provides a framework for a better understanding of structure–function relationships of this multifaceted primordial ribozyme.     

Design and isolation of ribozyme-substrate pairs using RNase P-based ribozymes containing altered substrate binding sites.

Substrate recognition and cleavage by the bacterial RNase P RNA requires two domains, a specificity domain, or S-domain, and a catalytic domain, or C-domain. The S-domain binds the T stem-loop region in a pre-tRNA substrate to confer specificity for tRNA substrates. In this work, the entire S-domain of the Bacillus subtilis RNase P RNA is replaced with an artificial substrate binding module. New RNA substrates are isolated by in vitro selection using two libraries containing random regions of 60 nt. At the end of the selection, the cleavage rates of the substrate library are approximately 0.7 min(-1)in 10 mM MgCl(2)at 37 degrees C, approximately 4-fold better than the cleavage of a pre-tRNA substrate by the wild-type RNase P RNA under the same conditions. The contribution of the S-domain replacement to the catalytic efficiency is from 6- to 22 000-fold. Chemical and nuclease mapping of two ribozyme-product complexes shows that this contribution correlates with direct interactions between the S-domain replacement and the selected substrate. These results demonstrate the feasibility of design and isolation of RNase P-based, matching ribozyme-substrate pairs without prior knowledge of the sequence or structure of the interactive modules in the ribozyme or substrate.     

The naturally trans-acting ribozyme RNase P RNA has leadzyme properties

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.     

Characterization of RNA sequence determinants and antideterminants of processing reactivity for a minimal substrate of Escherichia coli ribonuclease III

Members of the ribonuclease III family are the primary agents of double-stranded (ds) RNA processing in prokaryotic and eukaryotic cells. Bacterial RNase III orthologs cleave their substrates in a highly site-specific manner, which is necessary for optimal RNA function or proper decay rates. The processing reactivities of Escherichia coli RNase III substrates are determined in part by the sequence content of two discrete double-helical elements, termed the distal box (db) and proximal box (pb). A minimal substrate of E.coli RNase III, μR1.1 RNA, was characterized and used to define the db and pb sequence requirements for reactivity and their involvement in cleavage site selection. The reactivities of μR1.1 RNA sequence variants were examined in assays of cleavage and binding in vitro. The ability of all examined substitutions in the db to inhibit cleavage by weakening RNase III binding indicates that the db is a positive determinant of RNase III recognition, with the canonical UA/UG sequence conferring optimal recognition. A similar analysis showed that the pb also functions as a positive recognition determinant. It also was shown that the ability of the GC or CG bp substitution at a specific position in the pb to inhibit RNase III binding is due to the purine 2-amino group, which acts as a minor groove recognition antideterminant. In contrast, a GC or CG bp at the pb position adjacent to the scissile bond can suppress cleavage without inhibiting binding, and thus act as a catalytic antideterminant. It is shown that a single pb+db ‘set’ is sufficient to specify a cleavage site, supporting the primary function of the two boxes as positive recognition determinants. The base pair sequence control of reactivity is discussed within the context of new structural information on a post-catalytic complex of a bacterial RNase III bound to the cleaved minimal substrate.     

Dissecting functional cooperation among protein subunits in archaeal RNase P,a catalytic ribonucleoprotein complex

RNase P catalyzes the Mg²⁺-dependent 5′-maturation of precursor tRNAs. Biochemical studies on the bacterial holoenzyme, composed of one catalytic RNase P RNA (RPR) and one RNase P protein (RPP), have helped understand the pleiotropic roles (including substrate/Mg²⁺ binding) by which a protein could facilitate RNA catalysis. As a model for uncovering the functional coordination among multiple proteins that aid an RNA catalyst, we use archaeal RNase P, which comprises one catalytic RPR and at least four RPPs. Exploiting our previous finding that these archaeal RPPs function as two binary RPP complexes (POP5•RPP30 and RPP21•RPP29), we prepared recombinant RPP pairs from three archaea and established interchangeability of subunits through homologous/heterologous assemblies. Our finding that archaeal POP5•RPP30 reconstituted with bacterial and organellar RPRs suggests functional overlap of this binary complex with the bacterial RPP and highlights their shared recognition of a phylogenetically-conserved RPR catalytic core, whose minimal attributes we further defined through deletion mutagenesis. Moreover, single-turnover kinetic studies revealed that while POP5•RPP30 is solely responsible for enhancing the RPR’s rate of precursor tRNA cleavage (by 60-fold), RPP21•RPP29 contributes to increased substrate affinity (by 16-fold). Collectively, these studies provide new perspectives on the functioning and evolution of an ancient, catalytic ribonucleoprotein.     

Substrate Shape Preference of Escherichia coli Ribonuclease P Ribozyme and Holo Enzyme Using Bottom-Half Part-Shifting Variants of Pre-tRNA

We showed previously that the bacterial ribonuclease P (RNase P) ribozyme has substrate shape preference depending on the concentrations of catalytically important magnesium ions. The ribozyme discriminates a canonical cloverleaf precursor tRNA from a hairpin RNA with a CCA-tag sequence at low concentrations of magnesium ions. By detailed analysis of the shape preference using the bottom-half part-shifting variants of a tRNA precursor, we showed that the RNAs in a T-shape structure can be substrates for the ribozyme reactions even at low concentrations of magnesium ions, and that the RNA in a natural L-shape is the best substrate for both the ribozyme and the holo enzyme. The results also showed that the position of the bottom-half part did not affect the cleavage site selection of a substrate by the enzyme. Our results are the first kinetic evidence to show the importance of the bottom-half part of tRNA molecule, and our result also showed that the holo enzyme can discriminate substrate shape as well as the ribozyme at low concentrations of metal ions.