Effects of 5' leader and 3' trailer structures on pre-tRNA processing by nuclear RNase P

Eukaryotic transfer RNA precursors (pre-tRNAs) contain a 5' leader preceding the aminoacyl acceptor stem and a 3' trailer extending beyond this stem. An early step in pre-tRNA maturation is removal of the 5' leader by the endoribonuclease, RNase P. Extensive pairing between leader and trailer sequences has previously been demonstrated to block RNase P cleavage, suggesting that the 5' leader and 3' trailer sequences might need to be separated for the substrate to be recognized by the eukaryotic holoenzyme. To address whether the nuclear RNase P holoenzyme recognizes the 5' leader and 3' trailer sequences independently, interactions of RNase P with pre-tRNA(Tyr) containing either the 5' leader, the 3' trailer, or both were examined. Kinetic analysis revealed little effect of the 3' trailer or a long 5' leader on the catalytic rate (k(cat)) for cleavage using the various pre-tRNA derivatives. However, the presence of a 3' trailer that pairs with the 5' leader increases the K(m) of pre-tRNA slightly, in agreement with previous results. Similarly, competition studies demonstrate that removal of a complementary 3' trailer lowers the apparent K(I), consistent with the structure between these two sequences interfering with their interaction with the enzyme. Deletion of both the 5' and 3' extensions to give mature termini resulted in the least effective competitor. Further studies showed that the nuclear holoenzyme, but not the B. subtilis holoenzyme, had a high affinity for single-stranded RNA in the absence of attached tRNA structure. The data suggest that yeast nuclear RNase P contains a minimum of two binding sites involved in substrate recognition, one that interacts with tRNA and one that interacts with the 3' trailer. Furthermore, base pairing between the 5' leader and 3' trailer hinders recognition.     

Analysis of substrate recognition by the ribonucleoprotein endonuclease RNase P

Ribonuclease P (RNase P), is a ribonucleoprotein complex that catalyzes the site-specific cleavage of pre-tRNA and a wide variety of other substrates. Although RNase P RNA is the catalytic subunit of the holoenzyme, the protein subunit plays a critical role in substrate binding. Thus, RNase P is an excellent model system for studying ribonucleoprotein function. In this review we describe methods applied to the in vitro study of substrate recognition by bacterial RNase P, covering general considerations of reaction conditions, quantitative measurement of substrate binding equilibria, enzymatic and chemical protection, cross-linking, modification interference, and analysis of site-specific substitutions. We describe application of these methods to substrate binding by RNase P RNA alone and experimental considerations for examining the holoenzyme. The combined use of these approaches has shown that the RNA and protein subunits cooperate to bind different portions of the substrate structure, with the RNA subunit predominantly interacting with the mature domain of tRNA and the protein interacting with the 5(') leader sequence. However, important questions concerning the interface between the two subunits and the coordination of RNA and protein subunits in binding and catalysis remain.     

Recognition of the 5' leader of pre-tRNA substrates by the active site of ribonuclease P

The bacterial tRNA processing enzyme ribonuclease P (RNase P) is a ribonucleoprotein composed of a approximately 400 nucleotide RNA and a smaller protein subunit. It has been established that RNase P RNA contacts the mature tRNA portion of pre-tRNA substrates, whereas RNase P protein interacts with the 5' leader sequence. However, specific interactions with substrate nucleotides flanking the cleavage site have not previously been defined. Here we provide evidence for an interaction between a conserved adenosine, A248 in the Escherichia coli ribozyme, and N(-1), the substrate nucleotide immediately 5' of the cleavage site. Specifically, mutations at A248 result in miscleavage of substrates containing a 2' deoxy modification at N(-1). Compensatory mutations at N(-1) restore correct cleavage in both the RNA-alone and holoenzyme reactions, and also rescue defects in binding thermodynamics caused by A248 mutation. Analysis of pre-tRNA leader sequences in Bacteria and Archaea reveals a conserved preference for U at N(-1), suggesting that an interaction between A248 and N(-1) is common among RNase P enzymes. These results provide the first direct evidence for RNase P RNA interactions with the substrate cleavage site, and show that RNA and protein cooperate in leader sequence recognition.     

The affinity of magnesium binding sites in the Bacillus subtilis RNase P x pre-tRNA complex is enhanced by the protein subunit

The RNA subunit of bacterial ribonuclease P (RNase P) requires high concentrations of magnesium ions for efficient catalysis of tRNA 5'-maturation in vitro. The protein component of RNase P, required for cleavage of precursor tRNA in vivo, enhances pre-tRNA binding by directly contacting the 5'-leader sequence. Using a combination of transient kinetics and equilibrium binding measurements, we now demonstrate that the protein component of RNase P also facilitates catalysis by specifically increasing the affinities of magnesium ions bound to the RNase P x pre-tRNA(Asp) complex. The protein component does not alter the number or apparent affinity of magnesium ions that are either diffusely associated with the RNase P RNA polyanion or required for binding mature tRNA(Asp). Nor does the protein component alter the pH dependence of pre-tRNA(Asp) cleavage catalyzed by RNase P, providing further evidence that the protein component does not directly stabilize the catalytic transition state. However, the protein subunit does increase the affinities of at least four magnesium sites that stabilize pre-tRNA binding and, possibly, catalysis. Furthermore, this stabilizing effect is coupled to the P protein/5'-leader contact in the RNase P holoenzyme x pre-tRNA complex. These results suggest that the protein component enhances the magnesium affinity of the RNase P x pre-tRNA complex indirectly by binding and positioning pre-tRNA. Furthermore, RNase P is inhibited by cobalt hexammine (K(I) = 0.11 +/- 0.01 mM) while magnesium, manganese, cobalt, and zinc compete with cobalt hexammine to activate RNase P. These data are consistent with the hypothesis that catalysis by RNase P requires at least one metal-water ligand or one inner-sphere metal contact.     

The 5' leader of precursor tRNAAsp bound to the Bacillus subtilis RNase P holoenzyme has an extended conformation

RNase P catalyzes the 5' maturation of transfer RNA (tRNA). RNase P from Bacillus subtilis comprises a large RNA component (130 kDa, P RNA) and a small protein subunit (14 kDa, P protein). Although P RNA alone can efficiently catalyze the maturation reaction in vitro, P protein is strictly required under physiological conditions. We have used time-resolved fluorescence resonance energy transfer on a series of donor-labeled substrates and two acceptor-labeled P proteins to determine the conformation of the pre-tRNA 5' leader relative to the protein in the holoenzyme-pre-tRNA complex. The resulting distance distribution measurements indicate that the leader binds to the holoenzyme in an extended conformation between nucleotides 3 and 7. The conformational mobility of nucleotides 5-8 in the leader is reduced, providing further evidence that these nucleotides interact with the holoenzyme. The increased fluorescence intensity and lifetime of the 5'-fluorescein label of these leaders indicate a more hydrophobic environment, consistent with the notion that such interactions occur with the central cleft of the P protein. Taken together, our data support a model where the P protein binds to the 5' leader between the fourth and seventh nucleotides upstream of the cleavage site, extending the leader and decreasing its structural dynamics. Thus, P protein acts as a wedge to separate the 5' from the 3' terminus of the pre-tRNA and to position the cleavage site in the catalytic core. These results reveal a structural basis for the P protein dependent discrimination between precursor and mature tRNAs.     

Protein-Precursor tRNA Contact Leads to Sequence-Specific Recognition of 5′ Leaders by Bacterial Ribonuclease P

Bacterial ribonuclease P (RNase P) catalyzes the cleavage of 5′ leader sequences from precursor tRNAs (pre-tRNAs). Previously, all known substrate nucleotide specificities in this system are derived from RNA-RNA interactions with the RNase P RNA subunit. Here, we demonstrate that pre-tRNA binding affinities for Bacillus subtilis and Escherichia coli RNase P are enhanced by sequence-specific contacts between the fourth pre-tRNA nucleotide on the 5′ side of the cleavage site (N(− 4)) and the RNase P protein (P protein) subunit. B. subtilis RNase P has a higher affinity for pre-tRNA with adenosine at N(− 4), and this binding preference is amplified at physiological divalent ion concentrations. Measurements of pre-tRNA-containing adenosine analogs at N(− 4) indicate that specificity arises from a combination of hydrogen bonding to the N6 exocyclic amine of adenosine and steric exclusion of the N2 amine of guanosine. Mutagenesis of B. subtilis P protein indicates that F20 and Y34 contribute to selectivity at N(− 4). The hydroxyl group of Y34 enhances selectivity, likely by forming a hydrogen bond with the N(− 4) nucleotide. The sequence preference of E. coli RNase P is diminished, showing a weak preference for adenosine and cytosine at N(− 4), consistent with the substitution of Leu for Y34 in the E. coli P protein. This is the first identification of a sequence-specific contact between P protein and pre-tRNA that contributes to molecular recognition of RNase P. Additionally, sequence analyses reveal that a greater-than-expected fraction of pre-tRNAs from both E. coli and B. subtilis contains a nucleotide at N(− 4) that enhances RNase P affinity. This observation suggests that specificity at N(− 4) contributes to substrate recognition in vivo. Furthermore, bioinformatic analyses suggest that sequence-specific contacts between the protein subunit and the leader sequences of pre-tRNAs may be common in bacterial RNase P and may lead to species-specific substrate recognition.     

Binding and cleavage of unstructured RNA by nuclear RNase P

Ribonuclease P (RNase P) is an essential endoribonuclease for which the best-characterized function is processing the 5' leader of pre-tRNAs. Compared to bacterial RNase P, which contains a single small protein subunit and a large catalytic RNA subunit, eukaryotic nuclear RNase P is more complex, containing nine proteins and an RNA subunit in Saccharomyces cerevisiae. Consistent with this, nuclear RNase P has been shown to possess unique RNA binding capabilities. To understand the unique molecular recognition of nuclear RNase P, the interaction of S. cerevisiae RNase P with single-stranded RNA was characterized. Unstructured, single-stranded RNA inhibits RNase P in a size-dependent manner, suggesting that multiple interactions are required for high affinity binding. Mixed-sequence RNAs from protein-coding regions also bind strongly to the RNase P holoenzyme. However, in contrast to poly(U) homopolymer RNA that is not cleaved, a variety of mixed-sequence RNAs have multiple preferential cleavage sites that do not correspond to identifiable consensus structures or sequences. In addition, pre-tRNA(Tyr), poly(U)(50) RNA, and mixed-sequence RNA cross-link with purified RNase P in the RNA subunit Rpr1 near the active site in "Conserved Region I," although the exact positions vary. Additional contacts between poly(U)(50) and the RNase P proteins Rpr2p and Pop4p were identified. We conclude that unstructured RNAs interact with multiple protein and RNA contacts near the RNase P RNA active site, but that cleavage depends on the nature of interaction with the active site.     

The RNR motif of B. subtilis RNase P protein interacts with both PRNA and pre-tRNA to stabilize an active conformer

Ribonuclease P (RNase P) catalyzes the metal-dependent 5′ end maturation of precursor tRNAs (pre-tRNAs). In Bacteria, RNase P is composed of a catalytic RNA (PRNA) and a protein subunit (P protein) necessary for function in vivo. The P protein enhances pre-tRNA affinity, selectivity, and cleavage efficiency, as well as modulates the cation requirement for RNase P function. Bacterial P proteins share little sequence conservation although the protein structures are homologous. Here we combine site-directed mutagenesis, affinity measurements, and single turnover kinetics to demonstrate that two residues (R60 and R62) in the most highly conserved region of the P protein, the RNR motif (R60–R68 in Bacillus subtilis), stabilize PRNA complexes with both P protein (PRNA•P protein) and pre-tRNA (PRNA•P protein•pre-tRNA). Additionally, these data indicate that the RNR motif enhances a metal-stabilized conformational change in RNase P that accompanies substrate binding and is essential for efficient catalysis. Stabilization of this conformational change contributes to both the decreased metal requirement and the enhanced substrate recognition of the RNase P holoenzyme, illuminating the role of the most highly conserved region of P protein in the RNase P reaction pathway.     

Guide DNA technique reveals that the protein component of bacterial ribonuclease P is a modifier for substrate recognition

We developed a guide DNA technique with which the cleavage efficiency of pre-tRNA substrate raised in the RNase P reaction. The 20-mer guide DNAs hybridizing to the upstream region of the cleaving site enhanced the cleavage reactions of RNA substrates by Escherichia coli RNase P. This guide DNA technique was also applicable to cleavage site selection by choosing the DNA-hybridizing site. Results showed that RNase P accepts DNA/RNA double-stranded 5'-leader region with high catalytic efficiency as well as single-stranded RNA region in pre-tRNAs as substrates, which suggests that the protein component of bacterial RNase P prefers bulky nucleotides. The protein component did not affect the normal 5'-processing reaction of pre-tRNAs, but enhanced the mis-cleaving (hyperprocessing) reactions of tRNA in non-cloverleaf folding. Our results suggested that the protein component of RNase P is a modifier for substrate recognition.     

Pre-tRNA turnover catalyzed by the yeast nuclear RNase P holoenzyme is limited by product release

Ribonuclease P (RNase P) is a ribonucleoprotein that catalyzes the 5′ maturation of precursor transfer RNA in the presence of magnesium ions. The bacterial RNase P holoenzyme consists of one catalytically active RNA component and a single essential but catalytically inactive protein. In contrast, yeast nuclear RNase P is more complex with one RNA subunit and nine protein subunits. We have devised an affinity purification protocol to gently and rapidly purify intact yeast nuclear RNase P holoenzyme for transient kinetic studies. In pre-steady-state kinetic studies under saturating substrate concentrations, we observed an initial burst of tRNA formation followed by a slower, linear, steady-state turnover, with the burst amplitude equal to the concentration of the holoenzyme used in the reaction. These data indicate that the rate-limiting step in turnover occurs after pre-tRNA cleavage, such as mature tRNA release. Additionally, the steady-state rate constants demonstrate a large dependence on temperature that results in nonlinear Arrhenius plots, suggesting that a kinetically important conformational change occurs during catalysis. Finally, deletion of the 3′ trailer in pre-tRNA has little or no effect on the steady-state kinetic rate constants. These data suggest that, despite marked differences in subunit composition, the minimal kinetic mechanism for cleavage of pre-tRNA catalyzed by yeast nuclear RNase P holoenzyme is similar to that of the bacterial RNase P holoenzyme.     

Influence of Conformation of M. tuberculosis RNase P Protein Subunit on Its Function

RNase P is an essential enzyme that processes 5'' end leader sequence of pre-tRNA to generate mature tRNA. The bacterial RNase Ps contain a RNA subunit and one protein subunit, where the RNA subunit contains the catalytic activity. The protein subunit which lacks any catalytic activity, relaxes the ionic requirements for holoenzyme reaction and is indispensable for pre-tRNA cleavage in vivo. In the current study, we reconstituted the M. tuberculosis RNase P holoenzyme in vitro. We prepared the RNase P protein through two different strategies that differ in the conditions under which the recombinant M. tuberculosis protein, expressed in E. coli was purified. The mycobacterial RNase P protein which was purified under native conditions subsequent to isolation from inclusion bodies and in vitro renaturation, was capable of cleaving pre-tRNA specifically without the requirement of RNase P RNA. However, the preparation that was purified under denaturing conditions and refolded subsequently lacked any inherent pre-tRNA processing activity and cleaved the substrate only as a component of the holoenzyme with the RNA subunit. We found that the two RNase P protein preparations attained alternative conformations and differed with respect to their stability as well.     

A novel endonucleolytic mechanism to generate the CCA 3' termini of tRNA molecules in Thermotoga maritima

The tRNA 3'-terminal CCA sequence is essential for aminoacylation of the tRNAs and for translation on the ribosome. The tRNAs are transcribed as larger precursor molecules containing 5' and 3' extra sequences. In the tRNAs that do not have the encoded CCA, the 3' extra sequence after the discriminator nucleotide is usually cleaved off by the tRNA 3' processing endoribonuclease (3' tRNase, or RNase Z), and the 3'-terminal CCA residues are added thereto. Here we analyzed Thermotoga maritima 3' tRNase for enzymatic properties using various pre-tRNAs from T. maritima, in which all 46 tRNA genes encode CCA with only one exception. We found that the enzyme has the unprecedented activity that cleaves CCA-containing pre-tRNAs precisely after the CCA sequence, not after the discriminator. The assays for pre-tRNA variants suggest that the CA residues at nucleotides 75 and 76 are required for the enzyme to cleave pre-tRNAs after A at nucleotide 76 and that the cleavage occurs after nucleotide 75 if the sequence is not CA. Intriguingly, the pre-tRNA(Met) that is the only T. maritima pre-tRNA without the encoded CCA was cleaved after the discriminator. The kinetics data imply the existence of a CCA binding domain in T. maritima 3' tRNase. We also identified two amino acid residues critical for the cleavage site selection and several residues essential for the catalysis. Analysis of cleavage sites by 3' tRNases from another eubacteria Escherichia coli and two archaea Thermoplasma acidophilum and Pyrobaculum aerophilum corroborates the importance of the two amino acid residues for the cleavage site selection.     

Sequence changes in both flanking sequences of a pre-tRNA influence the cleavage specificity of RNase P.

The cleavage specificities of the RNase P holoenzymes from Escherichia coli and the yeast Schizosaccharomyces pombe and of the catalytic M1 RNA from E. coli were analyzed in 5'-processing experiments using a yeast serine pre-tRNA with mutations in both flanking sequences. The template DNAs were obtained by enzymatic reactions in vitro and transcribed with phage SP6 or T7 RNA polymerase. The various mutations did not alter the cleavage specificity of the yeast RNase P holoenzyme; cleavage always occurred predominantly at position G + 1, generating the typical seven base-pair acceptor stem. In contrast, the specificity of the prokaryotic RNase P activities, i.e. the catalytic M1 RNA and the RNase P holoenzyme from E. coli, was influenced by some of the mutated pre-tRNA substrates, which resulted in an unusual cleavage pattern, generating extended acceptor stems. The bases G - 1 and C + 73, forming the eighth base pair in these extended acceptor stems, were an important motif in promoting the unusual cleavage pattern. It was found only in some natural pre-tRNAs, including tRNA(SeCys) from E. coli, and tRNAs(His) from bacteria and chloroplasts. Also, the corresponding mature tRNAs in vivo contain an eight base pair acceptor stem. The presence of the CCA sequence at the 3' end of the tRNA moiety is known to enhance the cleavage efficiency with the catalytic M1 RNA. Surprisingly, the presence or absence of this sequence in two of our substrate mutants drastically altered the cleavage specificity of M1 RNA and of the E. coli holoenzyme, respectively. Possible reasons for the different cleavage specificities of the enzymes, the influence of sequence alterations and the importance of stacking forces in the acceptor stems are discussed.     

Eukaryotic RNase P: role of RNA and protein subunits of a primordial catalytic ribonucleoprotein in RNA-based catalysis

Ribonuclease P (RNase P) is an essential enzyme that processes the 5' leader sequence of precursor tRNA. Eubacterial RNase P is an RNA enzyme, while its eukaryotic counterpart acts as catalytic ribonucleoprotein, consisting of RNA and numerous protein subunits. To study the latter form, we reconstitute human RNase P activity, demonstrating that the subunits H1 RNA, Rpp21, and Rpp29 are sufficient for 5' cleavage of precursor tRNA. The reconstituted RNase P precisely delineates its cleavage sites in various substrates and hydrolyzes the phosphodiester bond. Rpp21 and Rpp29 facilitate catalysis by H1 RNA, which seems to require a phylogenetically conserved pseudoknot structure for function. Unexpectedly, Rpp29 forms a catalytic complex with M1 RNA of E. coli RNase P. The results uncover the core components of eukaryotic RNase P, reveal its evolutionary origin in translation, and provide a paradigm for studying RNA-based catalysis by other nuclear and nucleolar ribonucleoprotein enzymes.     

Novel mechanisms for maturation of chloroplast transfer RNA precursors

Despite the prokaryotic origins of chloroplasts, a plant chloroplast tRNA precursor is processed in a homologous in vitro system by a pathway distinct from that observed in Escherichia coli, but identical to that utilized for maturation of nuclear pre-tRNAs. The mature tRNA 5' terminus is generated by the site-specific endonucleolytic cleavage of an RNase P (or P-type) activity. The 3' end is likewise produced by a single precise endonucleolytic cut at the 3' terminus of the encoded tRNA domain. This is the first complete structural characterization of an organellar tRNA processing system using a homologous substrate. In contrast to eubacterial RNase P, chloroplast RNase P does not appear to contain an RNA subunit. The chloroplast activity bands with bulk protein at 1.28 g/ml in CsCI density gradients, whereas E.coli RNase P bands as ribonucleoprotein at 1.73 g/ml. Chloroplast RNase P activity survives treatment with micrococcal nuclease (MN) at levels 10- to 100-fold higher than those required to totally inactivate the E.coli enzyme. The chloroplast system is sensitive to a suppression of tRNA processing, caused by binding of inactive MN to pre-tRNA substrate, which is readily overcome by addition of carrier RNA to the assay.     

Probing the architecture of the B. subtilis RNase P holoenzyme active site by cross-linking and affinity cleavage

Bacterial ribonuclease P (RNase P) is a ribonucleoprotein complex composed of one catalytic RNA (PRNA) and one protein subunit (P protein) that together catalyze the 5' maturation of precursor tRNA. High-resolution X-ray crystal structures of the individual P protein and PRNA components from several species have been determined, and structural models of the RNase P holoenzyme have been proposed. However, holoenzyme models have been limited by a lack of distance constraints between P protein and PRNA in the holoenzyme-substrate complex. Here, we report the results of extensive cross-linking and affinity cleavage experiments using single-cysteine P protein variants derivatized with either azidophenacyl bromide or 5-iodoacetamido-1,10-o-phenanthroline to determine distance constraints and to model the Bacillus subtilis holoenzyme-substrate complex. These data indicate that the evolutionarily conserved RNR motif of P protein is located near (<15 Angstroms) the pre-tRNA cleavage site, the base of the pre-tRNA acceptor stem and helix P4 of PRNA, the putative active site of the enzyme. In addition, the metal binding loop and N-terminal region of the P protein are proximal to the P3 stem-loop of PRNA. Studies using heterologous holoenzymes composed of covalently modified B. subtilis P protein and Escherichia coli M1 RNA indicate that P protein binds similarly to both RNAs. Together, these data indicate that P protein is positioned close to the RNase P active site and may play a role in organizing the RNase P active site.     

Conformational change in the Bacillus subtilis RNase P holoenzyme–pre-tRNA complex enhances substrate affinity and limits cleavage rate

Ribonuclease P (RNase P) is a ribonucleoprotein complex that catalyzes the 5′ maturation of precursor tRNAs. To investigate the mechanism of substrate recognition in this enzyme, we characterize the thermodynamics and kinetics of Bacillus subtilis pre-tRNA^Asp binding to B. subtilis RNase P holoenzyme using fluorescence techniques. Time courses for fluorescein-labeled pre-tRNA binding to RNase P are biphasic in the presence of both Ca(II) and Mg(II), requiring a minimal two-step association mechanism. In the first step, the apparent bimolecular rate constant for pre-tRNA associating with RNase P has a value that is near the diffusion limit and is independent of the length of the pre-tRNA leader. Following formation of the initial enzyme–substrate complex, a unimolecular step enhances the overall affinity of pre-tRNA by eight- to 300-fold as the length of the leader sequence increases from 2 to 5 nucleotides. This increase in affinity is due to a decrease in the reverse rate constant for the conformational change that correlates with the formation of an optimal leader–protein interaction in the RNase P holoenzyme–pre-tRNA complex. Furthermore, the forward rate constant for the conformational change becomes rate limiting for cleavage under single-turnover conditions at high pH, explaining the origin of the observed apparent pK_a in the RNase P-catalyzed cleavage reaction. These data suggest that a conformational change in the RNase P•pre-tRNA complex is coupled to the interactions between the 5′ leader and P protein and aligns essential functional groups at the cleavage active site to enhance efficient cleavage of pre-tRNA.     

The inhibitory effect of the autoantigen La on in vitro 3' processing of mammalian precursor tRNAs

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can remove a 3' trailer from various precursor (pre)-tRNAs. We investigated what effect the autoantigen La has on 3' processing, since the La protein is known to bind to a 3'-terminal uridine tract of pre-tRNAs. We tested sixteen different pre-tRNA(Arg) substrates containing various 3' trailers with or without a 5' leader sequence for in vitro processing by pig 3' tRNase, and for gel-retardation in the presence or absence of human La protein. The R-TUUU series consists of four pre-tRNAs containing 6, 8, 11 and 15 nt 3' trailers ending with UUU and no 5' leader, while the R-TAGC series consists of the same four pre-tRNAs as R-TUUU except that the terminal sequence is AGC. The R-6LTUUU and R-6LTAGC series are derived from R-TUUU and R-TAGC, respectively, by adding a 6 nt 5' leader. La differentially inhibited their processing and bound to the pre-tRNAs; the 50 % inhibitory concentrations for the R-TUUU, R-TAGC, R-6LTUUU, and R-6LTAGC series were 82 to >850, >850, 2 to 292 and 573 to 785 nM, respectively, and the dissociation constants were 10 to 840, >850, 3 to 203 and 155 to 520 nM, respectively. These results indicate that both the terminal sequence UUU and the 5' leader contribute to more severe inhibition of 3' processing via tighter interaction with La. With respect to the R-TUUU and R-6LTUUU series, on the whole, the La inhibition was enhanced as the 3' trailer lengths decreased. Taken together, our results suggest that the La protein sterically hinders 3' tRNase from binding a pre-tRNA molecule probably near the cleavage site.     

A Divalent Cation Stabilizes the Active Conformation of the B. subtilis RNase P·Pre-tRNA Complex: A Role for an Inner-Sphere Metal Ion in RNase P

Metal ions interact with RNA to enhance folding, stabilize structure, and, in some cases, facilitate catalysis. Assigning functional roles to specifically bound metal ions presents a major challenge in analyzing the catalytic mechanisms of ribozymes. Bacillus subtilis ribonuclease P (RNase P), composed of a catalytically active RNA subunit (PRNA) and a small protein subunit (P protein), catalyzes the 5′-end maturation of precursor tRNAs (pre-tRNAs). Inner-sphere coordination of divalent metal ions to PRNA is essential for catalytic activity but not for the formation of the RNase P·pre-tRNA (enzyme-substrate, ES) complex. Previous studies have demonstrated that this ES complex undergoes an essential conformational change (to the ES^? conformer) before the cleavage step. Here, we show that the ES^? conformer is stabilized by a high-affinity divalent cation capable of inner-sphere coordination, such as Ca(II) or Mg(II). Additionally, a second, lower-affinity Mg(II) activates cleavage catalyzed by RNase P. Structural changes that occur upon binding Ca(II) to the ES complex were determined by time-resolved Förster resonance energy transfer measurements of the distances between donor-acceptor fluorophores introduced at specific locations on the P protein and pre-tRNA 5′ leader. These data demonstrate that the 5′ leader of pre-tRNA moves 4 to 6 Å closer to the PRNA·P protein interface during the ES-to-ES^? transition and suggest that the metal-dependent conformational change reorganizes the bound substrate in the active site to form a catalytically competent ES^? complex.