Multiple binding modes of substrate to the catalytic RNA subunit of RNase P from Escherichia coli.

M1 RNA that contained 4'-thiouridine was photochemically cross-linked to different substrates and to a product of the reaction it governs. The locations of the cross-links in these photochemically induced complexes were identified. The cross-links indicated that different substrates share some contacts but have distinct binding modes to M1 RNA. The binding of some substrates also results in a substrate-dependent conformational change in the enzymatic RNA, as evidenced by the appearance of an M1 RNA intramolecular cross-link. The identification of the cross-links between M1 RNA and product indicate that they are shared with only one of the three cross-linked E-S complexes that were identified, an indication of noncompetitive inhibition by the product. We also examined whether the cross-linked complexes between M1 RNA and substrate(s) or product are altered in the presence of the enzyme's protein cofactor (C5 protein) and in the presence of different concentrations of divalent metal ions. C5 protein enhanced the yield of certain M1 RNA-substrate cross-linked complexes for both wild-type M1 RNA and a deletion mutant of M1 RNA (delta[273-281]), but not for the M1 RNA-product complex. High concentrations of Mg2+ increased the yield of all M1 RNA-substrate complexes but not the M1 RNA-product complex.     

Important 2'-hydroxyl groups in model substrates for M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli.

The role of 2'-hydroxyl groups in a model substrate for RNase P from Escherichia coli was studied using mixed DNA/RNA derivatives of such a substrate. The presence of the 2'-hydroxyl groups of nucleotides at positions -1 and -2 in the leader sequence and at position 1, as well as at the first C in the 3'-terminal CCA sequence, are important but not absolutely essential for efficient cleavage of the substrate by RNase P or its catalytic RNA subunit, M1 RNA. The 2'-hydroxyl groups in the substrate that are important for efficient cleavage also participate in the binding of Mg2+. An all-DNA external guide sequence (EGS) can efficiently render a potential substrate, derived from the model substrate, susceptible to cleavage by the enzyme or its catalytic RNA subunit. Furthermore, both DNA and RNA EGSs turn over during the reaction with RNase P in vitro. The identity of the nucleotide at position 1 in the substrate, the adjacent Mg(2+)-binding site in the leader sequence, and the junction of the single and double-stranded regions are the important elements in the recognition of model substrates, as well as in the identification of the sites of cleavage in those model substrates.     

A novel tertiary interaction in M1 RNA, the catalytic subunit of Escherichia coli RNase P.

Phylogenetic covariation of the nucleotides corresponding to the bases at positions 121 and 236 in Escherichia coli RNase P RNA (M1 RNA) has been demonstrated in eubacterial RNase P RNAs. To investigate whether the nucleotides at these positions interact in M1 RNA we introduced base substitutions at either or at both of these positions. Single base substitutions at 121 or at 236 resulted in M1 RNA molecules which did not complement the temperature-sensitive phenotype associated with rnpA49 in vivo whereas wild-type M1 RNA or the double mutant M1 RNA, with restored base-pairing between 121 and 236, did. In addition, wild-type and the double mutant M1 RNA were efficiently cleaved by Pb++ between positions 122 and 123 whereas the rate of this cleavage was significantly reduced for the singly mutated M1 RNA variants. From these data we conclude that the nucleotides at positions 121 and 236 in M1 RNA establish a novel long-range tertiary interaction in M1 RNA. Our results also demonstrated that this interaction is not absolutely required for cleavage in vitro, however, a disruption resulted in a reduction in cleavage efficiency (kcat/Km), both in the absence and presence of C5.     

Protein synthesis inhibitors and catalytic RNA. Effect of puromycin on tRNA precursor processing by the RNA component of Escherichia coli RNase P

RNase P and ribosomes must interact with similar substrate molecules, tRNA precursors in the case of RNase P and aminoacyl-, peptidyl- or free tRNAs in the case of ribosomes. In order to compare the substrate recognition mechanisms between ribosomes and RNase P, protein synthesis inhibitors have been assayed for their effect on the catalytic activity of the RNA component of Escherichia coli RNase P (M1 RNA). Puromycin has an inhibitory effect that could be related to similar substrate recognition mechanisms by rRNA in the ribosome and by M1 RNA in RNase P.     

Product release is a rate-limiting step during cleavage by the catalytic RNA subunit of Escherichia coli RNase P.

The kinetic constants for cleavage of the tRNA(Tyr)Su3 precursor by the M1 RNA of E. coli RNase P were determined in the absence and presence of the C5 protein under single and multiple (steady state) turnover conditions. The rate constant of cleavage in the reaction catalyzed by M1 RNA alone was 5 times higher in single turnover than in multiple turnovers, suggesting that a rate-limiting step is product release. Cleavage by M1 RNA alone and by the holoenzyme under identical buffer conditions demonstrated that C5 facilitated product release. Addition of different product-like molecules under single turnover reaction conditions inhibited cleavage both in the absence and presence of C5. In the presence of C5, the Ki value for matured tRNA was approximately 20 times higher than in its absence, suggesting that C5 also reduces the interaction between the 5'-matured tRNA and the enzyme. In a growing cell the number of tRNA molecules is approximately 1000 times higher than the number of RNase P molecules. A 100-fold excess of matured tRNA over enzyme clearly inhibited cleavage in vitro. We discuss the possibility that RNase P is involved in the regulation of tRNA expression under certain growth conditions.     

G350 of Escherichia coli RNase P RNA contributes to Mg2+ binding near the active site of the enzyme

G350 of Escherichia coli RNase P RNA is a highly conserved residue among all bacteria and lies near the known magnesium binding site for the RNase P ribozyme, helix P4. Mutations at G350 have a dramatic effect on substrate cleavage activity for both RNA alone and holoenzyme; the G350C mutation has the most severe phenotype. The G350C mutation also inhibits growth of cells that express the mutant RNA in vivo under conditions of magnesium starvation. The results suggest that G350 contributes to Mg(2+) binding at helix P4 of RNase P RNA.     

Studies of the RNA degradosome-organizing domain of the Escherichia coli ribonuclease RNase E

The hydrolytic endoribonuclease RNase E, which is widely distributed in bacteria and plants, plays key roles in mRNA degradation and RNA processing in Escherichia coli. The enzymatic activity of RNase E is contained within the conserved amino-terminal half of the 118 kDa protein, and the carboxy-terminal half organizes the RNA degradosome, a multi-enzyme complex that degrades mRNA co-operatively and processes ribosomal and other RNA. The study described herein demonstrates that the carboxy-terminal domain of RNase E has little structure under native conditions and is unlikely to be extensively folded within the degradosome. However, three isolated segments of 10-40 residues, and a larger fourth segment of 80 residues, are predicted to be regions of increased structural propensity. The larger of these segments appears to be a protein-RNA interaction site while the other segments possibly correspond to sites of self-recognition and interaction with the other degradosome proteins. The carboxy-terminal domain of RNase E may thus act as a flexible tether of the degradosome components. The implications of these and other observations for the organization of the RNA degradosome are discussed.     

Base pairing between Escherichia coli RNase P RNA and its substrate.

Base pairing between the substrate and the ribozyme has previously been shown to be essential for catalytic activity of most ribozymes, but not for RNase P RNA. By using compensatory mutations we have demonstrated the importance of Watson-Crick complementarity between two well-conserved residues in Escherichia coli RNase P RNA (M1 RNA), G292 and G293, and two residues in the substrate, +74C and +75C (the first and second C residues in CCA). We suggest that these nucleotides base pair (G292/+75C and G293/+74C) in the ribozyme-substrate complex and as a consequence the amino acid acceptor stem of the precursor is partly unfolded. Thus, a function of M1 RNA is to anchor the substrate through this base pairing, thereby exposing the cleavage site such that cleavage is accomplished at the correct position. Our data also suggest possible base pairing between U294 in M1 RNA and the discriminator base at position +73 of the precursor. Our findings are also discussed in terms of evolution.     

The precursor tRNA 3'-CCA interaction with Escherichia coli RNase P RNA is essential for catalysis by RNase P in vivo

The L15 region of Escherichia coli RNase P RNA forms two Watson-Crick base pairs with precursor tRNA 3'-CCA termini (G292-C75 and G293-C74). Here, we analyzed the phenotypes associated with disruption of the G292-C75 or G293-C74 pair in vivo. Mutant RNase P RNA alleles (rnpBC292 and rnpBC293) caused severe growth defects in the E. coli rnpB mutant strain DW2 and abolished growth in the newly constructed mutant strain BW, in which chromosomal rnpB expression strictly depended on the presence of arabinose. An isosteric C293-G74 base pair, but not a C292-G75 pair, fully restored catalytic performance in vivo, as shown for processing of precursor 4.5S RNA. This demonstrates that the base identity of G292, but not G293, contributes to the catalytic process in vivo. Activity assays with mutant RNase P holoenzymes assembled in vivo or in vitro revealed that the C292/293 mutations cause a severe functional defect at low Mg2+ concentrations (2 mM), which we infer to be on the level of catalytically important Mg2+ recruitment. At 4.5 mM Mg2+, activity of mutant relative to the wild-type holoenzyme, was decreased only about twofold, but 13- to 24-fold at 2 mM Mg2+. Moreover, our findings make it unlikely that the C292/293 phenotypes include significant contributions from defects in protein binding, substrate affinity, or RNA degradation. However, native PAGE experiments revealed nonidentical RNA folding equilibria for the wild-type versus mutant RNase P RNAs, in a buffer- and preincubation-dependent manner. Thus, we cannot exclude that altered folding of the mutant RNAs may have also contributed to their in vivo defect.     

The catalytic RNA of RNase P from Escherichia coli cleaves Drosophila 2S ribosomal RNA in vitro: a new type of naturally occurring substrate for the ribozyme

The production of superoxide and nitric oxide induced in U87 glioma treated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) was examined by electron spin resonance (ESR) spectroscopy using a newly designed flow-type quartz cuvette without detaching cells from the culture plate. ESR spectra of 2,2,6, 6-tetramethyl-4-hydroxy-1-piperidinyloxy (TEMPOL) with U87 cells on a quartz culture plate were measured at 15 min intervals. The signal intensity of TEMPOL decreased in the presence of U87 cells at the pseudo-first order rate. The signal decay was accelerated in the U87 cells treated with LPS/IFN-gamma for 24 h, and was suppressed in the presence of superoxide dismutase and catalase. By the spin-trapping method, nitric oxide from U87 cells pretreated with LPS/IFN-gamma for 24 h was measured by the ESR, but only a weak signal of nitric oxide adducts was detected. Further, the nitrite and nitrate levels in the medium did not increase for 24 h. By the ESR measurement of cells on culture plates without detachment stress, it was found that the production of superoxide was induced by LPS/IFN-gamma, but that of nitric oxide was not, in U87 glioma cells.     

Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg²⁺-dependent cleavage of the 5′ leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250–500 mM Mg²⁺ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg²⁺ requirement to ∼10–20 mM and improves the rate and fidelity of cleavage. To understand the Mg²⁺- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPR_{C domain} and Cy5-RPR_{S domain}), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg²⁺-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg²⁺ cooperativity. Our findings highlight how Mg²⁺ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.     

Protein-RNA interactions in the RNase P holoenzyme from Escherichia coli

The genes for the protein (C5 protein) and RNA (M1 RNA) subunits of Escherichia coli RNase P have been subcloned and their products prepared in milligram quantities by rapid procedures. The interactions between the two subunits of the enzyme have been studied in vitro by a filter-binding technique. The stoichiometry of the subunits in the holoenzyme is 1:1. The dissociation constant for the specific interactions of the subunits in the holoenzyme complex is approximately 4 x 10(-10) M. C5 protein also interacts with various RNA molecules in a non-specific manner with a dissociation constant of 2 x 10(-8) to 6 x 10(-8) M. Regions of M1 RNA required for interaction with C5 protein have been defined by deletion analysis and footprinting techniques. These interactions are localized primarily between nucleotides 82 to 96 and 170 to 270 of M1 RNA.     

Guanosine 2-NH2 groups of Escherichia coli RNase P RNA involved in intramolecular tertiary contacts and direct interactions with tRNA.

We have identified by nucleotide analog interference mapping (NAIM) exocyclic NH2 groups of guanosines in RNase P RNA from Escherichia coli that are important for tRNA binding. The majority of affected guanosines represent phylogenetically conserved nucleotides. Several sites of interference could be assigned to direct contacts with the tRNA moiety, whereas others were interpreted as reflecting indirect effects on tRNA binding due to the disruption of tertiary contacts within the catalytic RNA. Our results support the involvement of the 2-NH2 groups of G292/G293 in pairing with C74 and C75 of tRNA CCA-termini, as well as formation of two consecutive base triples involving C75 and A76 of CCA-ends interacting with G292/A258 and G291/G259, respectively. Moreover, we present first biochemical evidence for two tertiary contacts (L18/P8 and L8/P4) within the catalytic RNA, whose formation has been postulated previously on the basis of phylogenetic comparative analyses. The tRNA binding interference data obtained in this and our previous studies are consistent with the formation of a consecutive nucleotide triple and quadruple between the tetraloop L18 and helix P8. Formation of the nucleotide triple (G316 and A94:U104 in wild-type E. coli RNase P RNA) is also supported by mutational analysis. For the mutant RNase P RNA carrying a G94:C104 double mutation, an additional G316-to-A mutation resulted in a restoration of binding affinity for mature and precursor tRNA.     

Monitoring the structure of Escherichia coli RNase P RNA in the presence of various divalent metal ions

Lead(II)-induced cleavage can be used as a tool to probe conformational changes in RNA. In this report, we have investigated the conformation of M1 RNA, the catalytic subunit of Escherichia coli RNase P, by studying the lead(II)-induced cleavage pattern in the presence of various divalent metal ions. Our data suggest that the overall conformation of M1 RNA is very similar in the presence of Mg(2+), Mn(2+), Ca(2+), Sr(2+) and Ba(2+), while it is changed compared to the Mg(2+)-induced conformation in the presence of other divalent metal ions, Cd(2+) for example. We also observed that correct folding of some M1 RNA domains is promoted by Pb(2+), while folding of other domain(s) requires the additional presence of other divalent metal ions, cobalt(III) hexamine or spermidine. Based on the suppression of Pb(2+) cleavage at increasing concentrations of various divalent metal ions, our findings suggest that different divalent metal ions bind with different affinities to M1 RNA as well as to an RNase P hairpin-loop substrate and yeast tRNA(Phe). We suggest that this approach can be used to obtain information about the relative binding strength for different divalent metal ions to RNA in general, as well as to specific RNA divalent metal ion binding sites. Of those studied in this report, Mn(2+) is generally among the strongest RNA binders.     

Mercurial-promoted Zn2+ release from Escherichia coli aspartate transcarbamoylase

The release of Zn2+ from aspartate transcarbamoylase (ATCase; c6r6) upon challenge by p-hydroxymercuriphenylsulfonate (PMPS) has been studied using the sensitive, high-affinity metallochromic indicator 4-(2-pyridylazo)resorcinol at pH 7.0. When the--SH group of each catalytic (c) chain is protected, 1 Zn2+ is released for every 4 eq of PMPS added to ATCase during titration of the 24--SH groups of regulatory (r) chains. Moreover, the release of Zn2+ is a linear function of PMPS added, indicating that the rate-limiting step in Zn2+ release is mercurial attack on the 1st of the 4 r--SH groups bonded tetrahedrally to Zn2+ in an r chain near c:r contacts. Dissociation of ATCase is linked to Zn2+ release and mercaptide formation; e.g. upon addition of 4 eq of PMPS to ATCase in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer, 1/6th of ATCase is dissociated to c3 and r2 subunits at approximately 83% of the rate of Zn2+ release, with no accumulation of the c6r4 intermediate as is observed in KPO4 buffer. Adding less than or equal to 4 PMPS/ATCase, the release of Zn2+ is first-order in [PMPS] and is virtually independent of [ATCase] with an activation energy of 18 kcal/mol. With large excesses of PMPS, stopped-flow traces show a lag period followed by pseudo first-order release of Zn2+ from ATCase and the reaction order in [PMPS] = approximately 1.3. Under these conditions, PMPS has a chaotropic effect on ATCase; the activation energy for Zn2+ release is much lower than that obtained with limiting PMPS and is increased by the presence of phosphate or active-site ligand from 6.6 to approximately 12 kcal/mol. A reasonable explanation of the observed kinetic data is that the organomercurial reagent binds reversibly to nitrogenous side chain groups in an ATCase molecule prior to the rate-limiting reaction with a sulfhydryl group.     

Importance of the +73/294 interaction in Escherichia coli RNase P RNA substrate complexes for cleavage and metal ion coordination

We have studied an interaction, the "73/294-interaction", between residues 294 in M1 RNA (the catalytic subunit of Escherichia coli RNase P) and +73 in the tRNA precursor substrate. The 73/294-interaction is part of the "RCCA-RNase P RNA interaction", which anchors the 3' R(+73)CCA-motif of the substrate to M1 RNA (interacting residues underlined). Considering that in a large fraction of tRNA precursors residue +73 is base-paired to nucleotide -1 immediately 5' of the cleavage site, formation of the 73/294-interaction results in exposure of the cleavage site. We show that the nature/orientation of the 73/294-interaction is important for cleavage site recognition and cleavage efficiency. Our data further suggest that this interaction is part of a metal ion-binding site and that specific chemical groups are likely to act as ligands in binding of Mg(2+) or other divalent cations important for function. We argue that this Mg(2+) is involved in metal ion cooperativity in M1 RNA-mediated cleavage. Moreover, we suggest that the 73/294-interaction operates in concert with displacement of residue -1 in the substrate to ensure efficient and correct cleavage. The possibility that the residue at -1 binds to a specific binding surface/pocket in M1 RNA is discussed. Our data finally rationalize why the preferred residue at position 294 in M1 RNA is U.     

Role of the sequence of the rne-dependent site in 3' processing of M1 RNA, the catalytic component of Escherichia coli RNase P

The 3' processing of M1 RNA, the catalytic component of Escherichia coli RNase P, occurs by two pathways involving multiple steps. The precursor of M1 RNA has an rne-dependent site downstream of the processing site, whose sequence variation affects the processing efficiency. In this study, we showed that the sequence itself of the rne-dependent site possessed the ability to determine the processing pathways and that it also affected the cleavage specificity with the generation of the processing products at one nucleotide upstream or downstream of the normal cleavage sites.     

Interaction between Escherichia coli RNase P RNA and the discriminator base results in slow product release.

We suggested previously that a purine at the discriminator base position in a tRNA precursor interacts with the well-conserved U294 in M1 RNA, the catalytic subunit of Escherichia coli RNase P. Here we investigated this interaction and its influence on the kinetics of cleavage as well as on cleavage site selection. The discriminator base in precursors to tRNA(Tyr)Su3 and tRNA(Phe) was changed from A to C and cleavage kinetics were studied by wild-type M1 RNA and a mutant M1 RNA carrying the compensatory substitution of a U to a G at position 294 in M1 RNA. Our data suggest that the discriminator base interacts with the residue at position 294 in M1 RNA. Although product release is a rate-limiting step both in the absence and in the presence of this interaction, its presence results in a significant reduction in the rate of product release. In addition, we studied cleavage site selection on various tRNA(His) precursor derivatives. These precursors carry a C at the discriminator base position. The results showed that the mutant M1 RNA harboring a G at position 294 miscleaved a wild-type tRNA(His) precursor and a tRNA(His) precursor carrying an inosine at the cleavage site. The combined data suggest a functional interaction between the discriminator base and the well-conserved U294 in M1 RNA. This interaction is suggested to play an important role in determining the rate of product release during multiple turnover cleavage of tRNA precursors by M1 RNA as well as in cleavage site selection.