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.     

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.     

Phylogenetic analysis of the structure of RNase MRP RNA in yeasts

RNase MRP is a ribonucleoprotein enzyme involved in processing precursor rRNA in eukaryotes. To facilitate our structure-function analysis of RNase MRP from Saccharomyces cerevisiae, we have determined the likely secondary structure of the RNA component by a phylogenetic approach in which we sequenced all or part of the RNase MRP RNAs from 17 additional species of the Saccharomycetaceae family. The structure deduced from these sequences contains the helices previously suggested to be common to the RNA subunit of RNase MRP and the related RNA subunit of RNase P, an enzyme cleaving tRNA precursors. However, outside this common region, the structure of RNase MRP RNA determined here differs from a previously proposed universal structure for RNase MRPs. Chemical and enzymatic structure probing analyses were consistent with our revised secondary structure. Comparison of all known RNase MRP RNA sequences revealed three regions with highly conserved nucleotides. Two of these regions are part of a helix implicated in RNA catalysis in RNase P, suggesting that RNase MRP may cleave rRNA using a similar catalytic mechanism.     

Identification of phosphates involved in catalysis by the ribozyme RNase P RNA.

The RNA subunit of ribonuclease P (RNase P RNA) is a catalytic RNA that cleaves precursor tRNAs to generate mature tRNA 5' ends. Little is known concerning the identity and arrangement of functional groups that constitute the active site of this ribozyme. We have used an RNase P RNA-substrate conjugate that undergoes rapid, accurate, and efficient self-cleavage in vitro to probe, by phosphorothioate modification-interference, functional groups required for catalysis. We identify four phosphate oxygens where substitution by sulfur significantly reduces the catalytic rate (50-200-fold). Interference at one site was partially rescued in the presence of manganese, suggesting a direct involvement in binding divalent metal ion cofactors required for catalysis. All sites are located in conserved sequence and secondary structure, and positioned adjacent to the substrate phosphate in a tertiary structure model of the ribozyme-substrate complex. The spatial arrangement of phosphorothioate-sensitive sites in RNase P RNA was found to resemble the distribution of analogous positions in the secondary and potential tertiary structures of other large catalytic RNAs.     

Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P

Ribonuclease P (RNase P) is an endonuclease that catalyzes the essential removal of the 5′ end of tRNA precursors. Until recently, all identified RNase P enzymes were a ribonucleoprotein with a conserved catalytic RNA component. However, the discovery of protein-only RNase P (PRORP) shifted this paradigm, affording a unique opportunity to compare mechanistic strategies used by naturally evolved protein and RNA-based enzymes that catalyze the same reaction. Here we investigate the enzymatic mechanism of pre-tRNA hydrolysis catalyzed by the NYN (Nedd4-BP1, YacP nuclease) metallonuclease of Arabidopsis thaliana, PRORP1. Multiple and single turnover kinetic data support a mechanism where a step at or before chemistry is rate-limiting and provide a kinetic framework to interpret the results of metal alteration, mutations, and pH dependence. Catalytic activity has a cooperative dependence on the magnesium concentration (n_H = 2) under k_cat/K_m conditions, suggesting that PRORP1 catalysis is optimal with at least two active site metal ions, consistent with the crystal structure. Metal rescue of Asp-to-Ala mutations identified two aspartates important for enhancing metal ion affinity. The single turnover pH dependence of pre-tRNA cleavage revealed a single ionization (pK_a ∼ 8.7) important for catalysis, consistent with deprotonation of a metal-bound water nucleophile. The pH and metal dependence mirrors that observed for the RNA-based RNase P, suggesting similar catalytic mechanisms. Thus, despite different macromolecular composition, the RNA and protein-based RNase P act as dynamic scaffolds for the binding and positioning of magnesium ions to catalyze phosphodiester bond hydrolysis.     

Binding of C5 Protein to P RNA Enhances the Rate Constant for Catalysis for P RNA Processing of Pre-tRNAs Lacking a Consensus G(+ 1)/C(+ 72) Pair

The RNA subunit of the ribonucleoprotein enzyme ribonuclease P (RNase P (P RNA) contains the active site, but binding of Escherichia coli RNase P protein (C5) to P RNA increases the rate constant for catalysis for certain pre-tRNA substrates up to 1000-fold. Structure-swapping experiments between a substrate that is cleaved slowly by P RNA alone (pre-tRNA^f-met605) and one that is cleaved quickly (pre-tRNA^met608) pinpoint the characteristic C(+ 1)/A(+ 72) base pair of initiator tRNA^f-met as the sole determinant of slow RNA-alone catalysis. Unlike other substrate modifications that slow RNA-alone catalysis, the presence of a C(+ 1)/A(+ 72) base pair reduces the rate constant for processing at both correct and miscleavage sites, indicating an indirect but nonetheless important role in catalysis. Analysis of the Mg²⁺ dependence of apparent catalytic rate constants for pre-tRNA^met608 and a pre-tRNA^met608 (+ 1)C/(+ 72)A mutant provides evidence that C5 promotes rate enhancement primarily by compensating for the decrease in the affinity of metal ions important for catalysis engendered by the presence of the CA pair. Together, these results support and extend current models for RNase P substrate recognition in which contacts involving the conserved (+ 1)G/C(+ 72) pair of tRNA stabilize functional metal ion binding. Additionally, these observations suggest that C5 protein has evolved to compensate for tRNA variation at positions important for binding to P RNA, allowing for tRNA specialization.     

Recent approaches to probe functional groups in ribonuclease P RNA by modification interference

Modification interference is a powerful method to identify important functional groups in RNA molecules. We review here recent developments of techniques to screen for chemical modifications that interfere with (i) binding of(pre-)tRNA to bacterial RNase P RNA or (ii) pre-tRNA cleavage by this ribozyme. For example, two studies have analyzed positions at which a substitution of sulfur for thepro-Rp oxygen affects tRNA binding [1] or catalysis [2]. The results emphasize the functional key role of a central core element present in all known RNase P RNA subunits. The four sulfur substitutions identified in one study [2] to inhibit the catalytic step also interfered with binding of tRNA toE. coli RNase P RNA [1]. This suggests that losses in binding energy due to the modification at these positions affect the enzyme-substrate and the enzyme-transition state complex. In addition, the two studies have revealed, for the first time, sites of direct metal ion coordination in RNase P RNA. The potentials, limitations and interpretational ambiguities of modification interference experiments as well as factors influencing their outcome are discussed.Abbreviations nt nucleotide(s) - PAGE polyacrylamide gel electrophoresis     

The catalytic core of RNase P.

A deletion mutant of the catalytic RNA component of Escherichia coli RNase P missing residues 87-241 retains the ability to interact with the protein component to form a functional catalyst. The deletion of this phylogenetically conserved region significantly increases the Km, indicating that the deleted structures may be important for binding to the precursor tRNA substrate but not for the cleavage reaction. Under some reaction conditions, this RNase P deletion mutant can become a relatively non-specific nuclease, indicating that this RNA's catalytic center may be more exposed. The catalytic core of the RNase P is formed by less than one third of the 377 residues of the RNase P RNA.     

NAIM and site-specific functional group modification analysis of RNase P RNA: magnesium dependent structure within the conserved P1-P4 multihelix junction contributes to catalysis

The tRNA processing endonuclease ribonuclease P contains an essential and highly conserved RNA molecule (RNase P RNA) that is the catalytic subunit of the enzyme. To identify and characterize functional groups involved in RNase P RNA catalysis, we applied self-cleaving ribozyme-substrate conjugates, on the basis of the RNase P RNA from Escherichia coli, in nucleotide analogue interference mapping (NAIM) and site-specific modification experiments. At high monovalent ion concentrations (3 M) that facilitate protein-independent substrate binding, we find that the ribozyme is largely insensitive to analogue substitution and that concentrations of Mg2+ (1.25 mM) well below that necessary for optimal catalytic rate (>100 mM) are required to produce interference effects because of modification of nucleotide bases. An examination of the pH dependence of the reaction rate at 1.25 mM Mg2+ indicates that the increased sensitivity to analogue interference is not due to a change in the rate-limiting step. The nucleotide positions detected by NAIM under these conditions are located exclusively in the catalytic domain, consistent with the proposed global structure of the ribozyme, and predominantly occur within the highly conserved P1-P4 multihelix junction. Several sensitive positions in J3/4 and J2/4 are proximal to a previously identified site of divalent metal ion binding in the P1-P4 element. Kinetic analysis of ribozymes with site-specific N7-deazaadenosine and deazaguanosine modifications in J3/4 was, in general, consistent with the interference results and also permitted the analysis of sites not accessible by NAIM. These results show that, in this region only, modification of the N7 positions of A62, A65, and A66 resulted in measurable effects on reaction rate and modification at each position displayed distinct sensitivities to Mg2+ concentration. These results reveal a restricted subset of individual functional groups within the catalytic domain that are particularly important for substrate cleavage and demonstrate a close association between catalytic function and metal ion-dependent structure in the highly conserved P1-P4 multihelix junction.     

RNase P: role of distinct protein cofactors in tRNA substrate recognition and RNA-based catalysis

The Escherichia coli ribonuclease P (RNase P) has a protein component, termed C5, which acts as a cofactor for the catalytic M1 RNA subunit that processes the 5′ leader sequence of precursor tRNA. Rpp29, a conserved protein subunit of human RNase P, can substitute for C5 protein in reconstitution assays of M1 RNA activity. To better understand the role of the former protein, we compare the mode of action of Rpp29 to that of the C5 protein in activation of M1 RNA. Enzyme kinetic analyses reveal that complexes of M1 RNA–Rpp29 and M1 RNA–C5 exhibit comparable binding affinities to precursor tRNA but different catalytic efficiencies. High concentrations of substrate impede the activity of the former complex. Rpp29 itself exhibits high affinity in substrate binding, which seems to reduce the catalytic efficiency of the reconstituted ribonucleoprotein. Rpp29 has a conserved C-terminal domain with an Sm-like fold that mediates interaction with M1 RNA and precursor tRNA and can activate M1 RNA. The results suggest that distinct protein folds in two unrelated protein cofactors can facilitate transition from RNA- to ribonucleoprotein-based catalysis by RNase P.     

Human tyrosine tRNA is also internally cleavable by E. coli ribonuclease P RNA ribozyme in vitro

Transfer RNA is an essential molecule for biological system, and each tRNA molecule commonly has a cloverleaf structure. Previously, we experimentally showed that some Drosophila tRNA (tRNA(Ala), tRNA(His), and tRNA(iMet)) molecules fit to form another, non-cloverleaf, structure in which the 3'-half of the tRNA molecules forms an alternative hairpin, and that the tRNA molecules are internally cleaved by the catalytic RNA of bacterial ribonuclease P (RNase P). Until now, the hyperprocessing reaction of tRNA has only been reported with Drosophila tRNAs. This time, we applied the hyperprocessing reaction to one of human tRNAs, human tyrosine tRNA, and we showed that this tRNA was also hyperprocessed by E. coli RNase P RNA. This tRNA is the first example for hyperprocessed non-Drosophila tRNAs. The results suggest that the hyperprocessing reaction can be a useful tool detect destablized tRNA molecules from any species.     

Catalysis by the RNA subunit of RNase P--a minireview

RNase P, an enzyme that contains both RNA and protein components, cleaves tRNA precursors to generate mature 5' termini. The catalytic activity of RNase P resides in the RNA component, with the protein cofactor affecting the rate of the cleavage reaction. The reaction is also influenced by the nature of the tRNA substrate.     

Evidence that binding of C5 protein to P RNA enhances ribozyme catalysis by influencing active site metal ion affinity

The RNA subunit (P RNA) of the bacterial RNase P ribonucleoprotein is a ribozyme that catalyzes the Mg-dependent hydrolysis of pre-tRNA, but it requires an essential protein cofactor (P protein) in vivo that enhances substrate binding affinities and catalytic rates in a substrate dependent manner. Previous studies of Bacillus subtilis RNase P, containing a Type B RNA subunit, showed that its cognate protein subunit increases the affinity of metal ions important for catalysis, but the functional role of these ions is unknown. Here, we demonstrate that the Mg2+ dependence of the catalytic step for Escherichia coli RNase P, which contains a more common Type A RNA subunit, is also modulated by its cognate protein subunit (C5), indicating that this property is fundamental to P protein. To monitor specifically the binding of active site metal ions, we analyzed quantitatively the rescue by Cd2+ of an inhibitory Rp phosphorothioate modification at the pre-tRNA cleavage site. The results show that binding of C5 protein increases the apparent affinity of the rescuing Cd2+, providing evidence that C5 protein enhances metal ion affinity in the active site, and thus is likely to contribute significantly to rate enhancement at physiological metal ion concentrations.     

Helix P4 is a divalent metal ion binding site in the conserved core of the ribonuclease P ribozyme

The ribonuclease P ribozyme (RNase P RNA), like other large ribozymes, requires magnesium ions for folding and catalytic function; however, specific sites of metal ion coordination in RNase P RNA are not well defined. To identify and characterize individual nucleotide functional groups in the RNase P ribozyme that participate in catalytic function, we employed self-cleaving ribozyme-substrate conjugates that facilitate measurement of the effects of individual functional group modifications. The self-cleavage rates and pH dependence of two different ribozyme-substrate conjugates were determined and found to be similar to the single turnover kinetics of the native ribozyme. Using site-specific phosphorothioate substitutions, we provide evidence for metal ion coordination at the pro-Rp phosphate oxygen of A67, in the highly conserved helix P4, that was previously suggested by modification-interference experiments. In addition, we detect a new metal ion coordination site at the pro-Sp phosphate oxygen of A67. These findings, in combination with the proximity of A67 to the pre-tRNA cleavage site, support the conclusion that an important role of helix P4 in the RNase P ribozyme is to position divalent metal ions that are required for catalysis.     

Studies on Escherichia coli RNase P RNA with Zn2+ as the catalytic cofactor

We demonstrate, for the first time, catalysis by Escherichia coli ribonuclease P (RNase P) RNA with Zn2+ as the sole divalent metal ion cofactor in the presence of ammonium, but not sodium or potassium salts. Hill analysis suggests a role for two or more Zn2+ ions in catalysis. Whereas Zn2+ destabilizes substrate ground state binding to an extent that precludes reliable Kd determination, Co(NH3)6(3+) and Sr2+ in particular, both unable to support catalysis by themselves, promote high-substrate affinity. Zn2+ and Co(NH3)6(3+) substantially reduce the fraction of precursor tRNA molecules capable of binding to RNase P RNA. Stimulating and inhibitory effects of Sr2+ on the ribozyme reaction with Zn2+ as cofactor could be rationalized by a model involving two Sr2+ ions (or two classes of Sr2+ ions). Both ions improve substrate affinity in a cooperative manner, but one of the two inhibits substrate conversion in a non-competitive mode with respect to the substrate and the Zn2+. A single 2'-fluoro modification at nt -1 of the substrate substantially weakened the inhibitory effect of Sr2+. Our results demonstrate that the studies on RNase P RNA with metal cofactors other than Mg2+ entail complex effects on structural equilibria of ribozyme and substrate RNAs as well as E*S formation apart from the catalytic performance.     

Human Tyrosine tRNA Is Also Internally Cleavable by E. coli Ribonuclease P RNA Ribozyme in Vitro

Transfer RNA is an essential molecule for biological system, and each tRNA molecule commonly has a cloverleaf structure. Previously, we experimentally showed that some Drosophila tRNA (tRNA^Ala, tRNA^His, and tRNA_i ^Met) molecules fit to form another, non-cloverleaf, structure in which the 3'-half of the tRNA molecules forms an alternative hairpin, and that the tRNA molecules are internally cleaved by the catalytic RNA of bacterial ribonuclease P (RNase P). Until now, the hyperprocessing reaction of tRNA has only been reported with Drosophila tRNAs. This time, we applied the hyperprocessing reaction to one of human tRNAs, human tyrosine tRNA, and we showed that this tRNA was also hyperprocessed by E. coli RNase P RNA. This tRNA is the first example for hyperprocessed non-Drosophila tRNAs. The results suggest that the hyperprocessing reaction can be a useful tool to detect destablized tRNA molecules from any species.     

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.     

The protein component of bacterial ribonuclease P flickers the metal ion response to the substrate shape preference of the ribozyme

The substrate shape specificity of the Escherichia coli ribonuclease P (RNase P) ribozyme depends on the concentration of magnesium ion. At 10 mM or more, it can cleave a hairpin substrate as well as a cloverleaf pre-transfer RNA (tRNA). The results showed, however, that the holo enzyme cleaved the hairpin substrate at low concentrations of magnesium ion. Considering that the homologous E. coli tRNAs are resistant to internal cleavage by the RNase P, the phenomena suggest that this catalytic activity might take part in the removing the mis-folded RNAs in the cell.     

Evidence for induced fit in bacterial RNase P RNA-mediated cleavage

RNase P with its catalytic RNA subunit is involved in the processing of a number of RNA precursors with different structures. However, precursor tRNAs are the most abundant substrates for RNase P. Available data suggest that a tRNA is folded into its characteristic structure already at the precursor state and that RNase P recognizes this structure. The tRNA D-/T-loop domain (TSL-region) is suggested to interact with the specificity domain of RNase P RNA while residues in the catalytic domain interact with the cleavage site. Here, we have studied the consequences of a productive interaction between the TSL-region and its binding site (TBS) in the specificity domain using tRNA precursors and various hairpin-loop model substrates. The different substrates were analyzed with respect to cleavage site recognition, ground-state binding, cleavage as a function of the concentration of Mg(2+) and the rate of cleavage under conditions where chemistry is suggested to be rate limiting using wild-type Escherichia coli RNase P RNA, M1 RNA, and M1 RNA variants with structural changes in the TBS-region. On the basis of our data, we conclude that a productive TSL/TBS interaction results in a conformational change in the M1 RNA substrate complex that has an effect on catalysis. Moreover, it is likely that this conformational change comprises positioning of chemical groups (and Mg(2+)) at and in the vicinity of the cleavage site. Hence, our findings are consistent with an induced-fit mechanism in RNase P RNA-mediated cleavage.