Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.26.5 (RNase P)
1,348 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A complex pseudoknot structure surrounds the first ribosome initiation site in the Escherichia coli alpha mRNA and mediates its regulation by ribosomal protein S4. A 112 nt RNA fragment containing this pseudoknot exists in two conformations that are resolvable by gel electrophoresis below room temperature. Between 30 degrees C and 45 degrees C the conformers reach thermodynamic equilibrium on a time scale ranging from one hour to one minute, and the interconversion between conformers is linked to H+, K+ and Mg2+ concentrations. Mg2+ favors formation of the "fast" electrophoretic form: a single Mg2+ is bound in the rate-limiting step, followed by cooperative binding of approximately 1.7 additional ions. Binding of the latter ions provides most of the favorable free energy for the reaction. However, the "slow" form binds about the same number of Mg ions, albeit more weakly, so that saturating Mg2+ concentrations drive the equilibrium to only approximatley 70% fast form. A single H+ is taken up in the switch to the "slow" conformer, which has apparent pK approximately 5.9; low pH also stabilizes part of the pseudoknot structure melting at approximately 62 degrees C. Mg2+ and H+ appear to direct alpha mRNA folding by relatively small (10 to 100-fold) differences in their affinities for alternative conformers. K+ has very little effect on the conformational equilibrium, but at high concentrations accelerates interconversion between the conformers. The alpha mRNA conformational switch is similar in its slow kinetics, large activation energy, and Mg2+ dependence of the equilibrium constant to slow steps in the folding of tRNA, group I introns, and RNase P RNA tertiary structures, though it differs from these in the association of a single Mg2+ with the rate-limiting step.
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PMID:Effects of Mg2+, K+, and H+ on an equilibrium between alternative conformations of an RNA pseudoknot. 923 10

The folding thermodynamics and kinetics for the ribozyme from Bacillus subtilis RNase P are analyzed using circular dichroism and UV absorbance spectroscopies and catalytic activity. At 37 degrees C, the addition of Mg2+ (Kd approximately 50 microM) to the unfolded state produces an intermediate state within 1 ms which contains a comparable amount of secondary structure as the native ribozyme. The subsequent transition to the native state (Kd[Mg] approximately 0.8 mM, Hill coefficient approximately 3.5) has a half-life of hundreds of seconds as measured by circular dichroism at 278 nm and by a ribozyme activity assay. Surprisingly, the formation of the native structure is accelerated strongly by the addition of a denaturant; approximately 30-fold at 4.5 M urea. Thus, the rate-limiting step entails the disruption of a considerable number of interactions. The folding of this, and presumably other large RNAs, is slow due to the structural rearrangement of kinetically trapped species. Taken together with previous submillisecond relaxation kinetics of tRNA tertiary structure, we suggest that error-free RNA folding can be on the order of milliseconds.
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PMID:Intermediates and kinetic traps in the folding of a large ribozyme revealed by circular dichroism and UV absorbance spectroscopies and catalytic activity. 936 Jun 10

The ribozyme RNase P absolutely requires divalent metal ions for catalytic function. Multiple Mg2+ ions contribute to the optimal catalytic efficiency of RNase P, and it is likely that the tertiary structure of the ribozyme forms a specific metal-binding pocket for these ions within the active-site. To identify base moieties that contribute to catalytic metal-binding sites, we have used in vitro selection to isolate variants of the Escherichia coli RNase P RNA with altered specificities for divalent metal. RNase P RNA variants with increased activity in Ca2+ were enriched over 18 generations of selection for catalysis in the presence of Ca2+, which is normally disfavored relative to Mg2+. Although a wide spectrum of mutations was found in the generation-18 clones, only a single point mutation was common to all clones: a cytosine-to-uracil transition at position 70 (E. coli numbering) of RNase P. Analysis of the C70U point mutant in a wild-type background confirmed that the identity of the base at position 70 is the sole determinant of Ca2+ selectivity. It is noteworthy that C70 lies within the phylogenetically well conserved J3/4-P4-J2/4 region, previously implicated in Mg2+ binding. Our finding that a single base change is sufficient to alter the metal preference of RNase P is further evidence that the J3/4-P4-J2/4 domain forms a portion of the ribozyme's active site.
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PMID:In vitro selection for altered divalent metal specificity in the RNase P RNA. 940 16

Ribonuclease P (RNase P) contains a catalytic RNA that cleaves precursor tRNA (pre-tRNA) to form the mature 5'-end of tRNA. Previous kinetic analyses with mutant pre-tRNAs indicated that both C residues of the invariant 3'-terminal CCA form specific interactions with RNase P RNA that contribute to the energetics of substrate binding (1, 2). In the present study, we have used single-turnover kinetic analysis to investigate whether specific changes in the 3'-terminal CCA influence the rate of the chemical step through which enzyme-bound substrate is converted to product (k2). At optimal ionic strength (1.0 M NH4Cl, 25 mM MgCl2), deletion or substitution of the 3'-proximal C residue (CCA) reduced the rate of the chemical step of cleavage (k2) by 60-fold. Similar changes to the 5'-proximal C residue (CCA) or the 3'-terminal A residue (CCA) reduced k2 only a few fold. Each mutant substrate exhibited weakened affinity for Mg2+, as measured by Hill plots, and the severity of these defects correlated with the observed reductions in k2. Furthermore, elevated concentrations of Mg2+ partially, but not completely, suppress the k2 defects caused by deletion or substitution of the 3'-proximal C residue. We conclude that the 3'-CCA of pre-tRNA, particularly the 3'-proximal C residue, comprises part of the catalytic pocket formed in the pre-tRNA-RNase P complex and participates in the binding of Mg2+ ions that are essential for catalysis by RNase P RNA.
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PMID:Participation of the 3'-CCA of tRNA in the binding of catalytic Mg2+ ions by ribonuclease P. 958 41

We have studied the structure and divalent metal ion binding of a domain of the ribozyme RNase P RNA that is involved in base pairing with its substrate. Our data suggest that the folding of this internal loop, the P15-loop, is similar irrespective of whether it is part of the full-length ribozyme or part of a model RNA molecule. We also conclude that this element constitutes an autonomous divalent metal ion binding domain of RNase P RNA and our data suggest that certain specific chemical groups within the P15-loop participate in coordination of divalent metal ions. Substitutions of the Sp- and Rp-oxygens with sulfur at a specific position in this loop result in a 2.5-5-fold less active ribozyme, suggesting that Mg2+ binding at this position contributes to function. Our findings strengthen the concept that small RNA building blocks remain basically unchanged when removed from their structural context and thus can be used as models for studies of their potential function and structure within native RNA molecules.
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PMID:The P15-loop of Escherichia coli RNase P RNA is an autonomous divalent metal ion binding domain. 967 Oct 51

The ribonucleoprotein ribonuclease P (RNase P) cleaves all tRNA precursors endonucleolitically to produce the mature 5'-end. Dictyostelium discoideum RNase P displays an absolute requirement for Mg2+. Only the alkaline earth cations Ca2+, Sr2+, and Ba2+, under appropriate conditions can substitute to some extent for Mg2+. The transition metals Mn2+, Co2+, Ni2+, and Cd2+ are efficient inhibitors of the enzyme activity. Ca2+, Sr2+ and Ba2+, in the presence of Mg2+, exhibit a bimodal action at the kinetic phase of the reaction. Kinetic analysis of the activation phase revealed that Ca2+, Sr2+, or Ba2+ attached on a specific site of RNase P act as nonessential-noncompetitive activators. Further additions of Ca2+, Sr2+, or Ba2+ cause noncompetitive inhibition on the RNase P reaction, indicating that RNase P possesses a second binding site responsible for the inhibitory effect of Ca2+, Sr2+, and Ba2+. Both activator and inhibitory sites can be occupied by Ca2+, Sr2+, or Ba2+ at the same time.
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PMID:Bimodal action of alkaline earth cations on Dictyostelium discoideum ribonuclease P activity. 979 10

The thermodynamics and folding kinetics of a circularly permuted construct of the ribozyme from Bacillus subtilis RNase P are analyzed and compared with the folding properties of the wild-type ribozyme using optical spectroscopy and catalytic activity. The folding of the wild-type ribozyme is slow due to the rearrangement of kinetically trapped species containing misfolded structures. To test whether any misfolded structure arises from interactions between the two independently folding domains of the RNase P RNA, a circular permuted form was created where one of the two phosphodiester bonds connecting these domains is broken. This construct folds approximately 15-fold faster (t1/2 approximately nine seconds) than the wild-type ribozyme at 37 degreesC. While the complete folding of both domains is kinetically indistinguishable in the wild-type ribozyme, one domain folds much faster than the other domain in the circularly permuted construct. Hence, the major kinetic trap in the folding of the wild-type RNase P RNA involves interdomain interactions. This kinetic trap is avoidable at 37 degreesC in the circularly permuted RNA. However, at temperatures below 30 degreesC or when refolding begins from an equilibrium intermediate stabilized by submillimolar concentrations of Mg2+, a subpopulation containing an interdomain misfold still forms. These results indicate that the folding pathway of this large RNA is highly malleable and can be under kinetic control.
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PMID:Pathway modulation, circular permutation and rapid RNA folding under kinetic control. 1002 46

A number of aminoglycosides have been reported to interact and interfere with the function of various RNA molecules. Among these are 16S rRNA, the group I intron, and the hammerhead ribozymes. In this report we show that cleavage by RNase P RNA in the absence as well as in the presence of the RNase P protein is inhibited by several aminoglycosides. Among the ones we tested, neomycin B was found to be the strongest inhibitor with a Ki value in the micromolar range (35 microM). Studies of lead(II)-induced cleavage of RNase P RNA suggested that binding of neomycin B interfered with the binding of divalent metal ions to the RNA. Taken together, our findings suggest that aminoglycosides compete with Mg2+ ions for functionally important divalent metal ion binding sites. Thus, RNase P, which is an essential enzyme, is indeed a potential drug target that can be used to develop new drugs by using various aminoglycosides as lead compounds.
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PMID:Inhibition of RNase P RNA cleavage by aminoglycosides. 1033 57

Precursor tRNA (ptRNA) substrates carrying a single Rp or Sp-phosphorothioate modification at the RNase P cleavage site were used as tools to study the cleavage mechanism of RNase P RNA from Bacillus subtilis. Both the Sp and the Rp-diastereomer reduced the rate of processing at least 10(4)-fold under conditions where the chemical step is essentially rate-limiting. Neither the Rp nor the Sp-phosphorothioate modification affected ptRNA ground state binding to B. subtilis RNase P RNA. Processing of the Rp-diastereomeric ptRNA could be restored in the presence of Mn2+or Cd2+, demonstrating direct metal ion coordination to the pro -Rp oxygen during catalysis. With Cd2+, processing required the presence of another metal ion, such as Ca2+or Mg2+, to mediate substrate binding. This is in contrast to Escherichia coli RNase P RNA, which promotes cleavage of Rp-diastereomeric ptRNA in the presence of Cd2+as the sole divalent metal ion. Analysis of [Cd2+]-dependent processing of the Rp-diastereomeric substrate by B. subtilis RNase P RNA was consistent with the involvement of at least two metal ions in catalysis. The presence of two catalytic metal ion binding sites is also supported by the inhibition mode of Ca2+on cleavage of unmodified ptRNA. In the presence of an Sp-phosphorothioate modification at the scissile bond, neither Mn2+nor Cd2+were able to restore significant cleavage at this location. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Unaffected ground state binding of the Sp-diastereomeric ptRNA but a >/=10(4)-fold reduced hydrolysis rate may indicate a crucial role of the pro -Sp oxygen in transition state stabilization or may be attributed to steric exclusion of catalytic metal ions. Based on our comparative analyses of B. subtilis and E. coli RNase P RNA, each representing the main structural subtypes of bacterial RNase P RNA, common features in terms of active site constraints and role of catalytic metal ions can now be formulated for bacterial RNase P RNAs. On the other hand, substantial and unexpected differences with respect to the overall metal ion requirements and tRNA binding modes have been observed for the two catalytic RNAs.
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PMID:Role of metal ions in the hydrolysis reaction catalyzed by RNase P RNA from Bacillus subtilis. 1039 Mar 42

The cleavage step of bacterial RNase P catalysis involves concentration-independent processes after the formation of the ribozyme-substrate complex that result in the breaking of a phosphodiester bond. The 2'OH group at the cleavage site of a pre-tRNA substrate is an important determinant in the cleavage step. We determined here that in contrast to a tRNA substrate, the 2'OH at the cleavage site of two in vitro selected substrates has no effect, whereas a 2'OH located adjacent to the cleavage site has a similarly large effect on the cleavage step. This result indicates that a unique 2'OH in the vicinity of the cleavage site interacts with the ribozyme to achieve the maximal efficiency of the cleavage step. Individual modifications in a pre-tRNA substrate that disrupt ES interactions proximal to the cleavage site generally have little effect on the usage of this unique 2'OH. Ribozyme modifications that delete the interactions involving the T stem-loop of the tRNA have a large effect on the usage of this unique 2'OH and also alter the location of this 2'OH. We propose a new ES complex prior to the bond-breaking step in the reaction scheme to explain these results. This second ES complex is in fast equilibrium with the initial ES complex formed by bimolecular collision. The ribozyme interaction with this unique 2'OH shifts the equilibrium in favor of the second ES complex. The formation of the second ES complex may require optimal geometry of the two independently folding domains of this ribozyme to precisely position crucial functional groups and Mg2+ ions in the active site. Such a domain geometry is significantly favored by the RNase P protein. In the absence of the protein, spatial rearrangement of these domains in the ES complex may be necessary.
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PMID:The cleavage step of ribonuclease P catalysis is determined by ribozyme-substrate interactions both distal and proximal to the cleavage site. 1039 36


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