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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)

Our results indicate that RNase P has a very general role in the processing of tRNA precursors in E. coli, being responsible for the cleavage of virtually all precursor molecules at a site corresponding to the 5' end of the mature tRNA, and that at least two other RNases play specific roles in precursor processing. One of these, which may be RNase II, is responsible for removing extra nucleotides from the 3' end of tRNA precursors. The other, which we call RNase P2, is an endonuclease that cleaves precursors in spacer regions between different tRNA sequences; this enzyme is involved in the processing of large multimeric precursors.
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PMID:Processing of E. coli tRNA precursors. 110

tRNA affinity chromatography, based on complex formation between tRNAs with complementary anticodons, has been applied to the isolation of specific tRNA precursors. When [32P]RNA, isolated from an Escherichia coli strain containing a thermolabile ribonuclease P, was chromatographed on resin-bound yeast phenylalanine tRNA, precursor tRNAGlu (possessing the complementary anticodon) was specifically retained. Likewise, precursor tRNAPhe was isolated from a column of resin-bound E. coli glutamate tRNA. Both precursor tRNAs isolated were monomeric and may be processed products of an originally larger RNA precursor. Both tRNA precursors contain additional nucleotides beyond the 5'-end of the mature tRNA and have all modified bases found in mature tRNA. The method can be extended to isolate other tRNA precursors by affinity chromatography with different tRNAs. Since the principle of complementary anticodon interaction is not restricted to any particular organism, specific precursor tRNAs from other sources may also be isolated in this way.
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PMID:A method for the isolation of specific tRNA precursors. 110 1

The RNase P cleavage reaction was studied as a function of the number of base-pairs in the acceptor-stem and/or T-stem of a natural tRNA precursor, the tRNA(Tyr)Su3 precursor. Our data suggest that the location of the Escherichia coli RNase P cleavage site does not depend merely on the lengths of the acceptor-stem and T-stem as previously suggested. Surprisingly, we find that precursors with only four base-pairs in the acceptor-stem are cleaved by M1 RNA and by holoenzyme. Furthermore, we show that both disruption of base-pairing, and alteration of the nucleotide sequence (without disruption of base-pairing) proximal to the cleavage site result in aberrant cleavage. Thus, the identity of the nucleotides near the cleavage site is important for recognition of the cleavage site rather than base-pairing. The important nucleotides are those at positions -2, -1, +1, +72, +73 and +74. We propose that the nucleotide at position +1 functions as a guiding nucleotide. These results raise the possibility that Mg2+ binding near the cleavage site is dependent on the identity of the nucleotides at these positions. In addition, we show that disruption of base-pairing in the acceptor-stem affects both Michaelis-Menten constants, Km and kcat.
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PMID:Several regions of a tRNA precursor determine the Escherichia coli RNase P cleavage site. 127 79

We compared cleavage efficiencies of mono-molecular and bipartite model RNAs as substrates for RNase P RNAs (M1 RNAs) and holoenzymes from E. coli and Thermus thermophilus, an extreme thermophilic eubacterium. Acceptor stem and T arm of pre-tRNA substrates are essential recognition elements for both enzymes. Impairing coaxial stacking of acceptor and T stems and omitting the T loop led to reduced cleavage efficiencies. Small model substrates were less efficiently cleaved by M1 RNA and RNase P from T. thermophilus than by the corresponding E. coli activities. Competition kinetics and gel retardation studies showed that truncated tRNA substrates are less tightly bound by RNase P and M1 RNA from both bacteria. Our data further indicate that (pre-)tRNA interacts stronger with E. coli than T. thermophilus M1 RNA. Thus, low cleavage efficiencies of truncated model substrates by T. thermophilus RNase P or M1 RNA could be explained by a critical loss of important contact points between enzyme and substrate. In addition, acceptor stem--T arm substrates, composed of two synthetic RNA fragments, have been designed to mimic internal cleavage of any target RNA molecule available for base pairing.
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PMID:Cleavage efficiencies of model substrates for ribonuclease P from Escherichia coli and Thermus thermophilus. 128 15

The genes encoding the RNA subunit of ribonuclease P from the unicellular cyanobacterium Synechocystis sp. PCC 6803, and from the heterocyst-forming strains Anabaena sp. PCC 7120 and Calothrix sp. PCC 7601 were cloned using the homologous gene from Anacystis nidulans (Synechococcus sp. PCC 6301) as a probe. The genes and the flanking regions were sequenced. The genes from Anabaena and Calothrix are flanked at their 3'-ends by short tandemly repeated repetitive (STRR) sequences. In addition, two other sets of STRR sequences were detected within the transcribed regions of the Anabaena and Calothrix genes, increasing the length of a variable secondary structure element present in many RNA subunits of ribonuclease P from eubacteria. The ends of the mature RNAs were determined by primer extension and RNase protection. The predicted secondary structure of the three RNAs studied is similar to that of Anacystis and although some idiosyncrasies are observed, fits well with the eubacterial consensus.
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PMID:Analysis of the gene encoding the RNA subunit of ribonuclease P from cyanobacteria. 128 40

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme which participates in processing precursor tRNAs. The RNA subunit contains the catalytic site and is capable of catalysis in the absence of the protein subunit. RNase P RNAs from various eubacteria consist of a core of conserved sequence and secondary structure which is evolutionarily modified in different organisms by the presence of discrete helical elements at various sites in the RNAs. The variable occurrence of these helical elements suggests that they have no important functional role in the enzyme. The Escherichia coli RNase P RNA contains four such elements. It has been shown that simultaneous deletion of all four of them produces an RNA that is functional but has several significant defects which could arise from general disruption of the RNA or from the loss of element-specific functions. This paper describes a more detailed analysis of the role of the variable elements in E. coli RNase P RNA. Removal of one of the elements had no apparent effect on RNase P activity in vitro. Two other elements are required for correct folding of the RNA: their absence confers a requirement for extremely high monovalent salt concentrations, apparently to reduce intramolecular electrostatic repulsion. The fourth element that was tested participates in a long-range structural interaction (pseudoknot) which contributes to the structural stability of the enzyme and affects substrate binding affinity. In the absence of this helix, the RNA becomes temperature-sensitive, and the KM increases 100-fold.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Contributions of phylogenetically variable structural elements to the function of the ribozyme ribonuclease P. 137 Jun 27

A high yield, photoactivated cross-linking reaction between a modified tRNA and RNase P RNA was used as a quantitative assay of substrate binding affinity. The cross-linking assay allows the effects of metal ions on substrate binding to be measured independently and in the absence of the pre-tRNA cleavage reaction. The results of this assay, in conjunction with the conventional cleavage assay, support the following conclusions about the nature of the RNase P RNA-tRNA binding interaction. (i) Monovalent cations act primarily to enhance enzyme-substrate binding, presumably by functioning as counterions. This enhancement can be attributed to a reduction in the tRNA off-rate. (ii) Although divalent cation is required for cleavage, the enzyme-substrate complex can form in the absence of divalent cation; the essential role of divalent cation in the reaction is thus catalytic. (iii) Ca2+ is as efficient as Mg2+ in promoting binding but supports catalysis only at a low rate.
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PMID:Influence of metal ions on the ribonuclease P reaction. Distinguishing substrate binding from catalysis. 137 Aug 19

As the result of an unusual RNase P specificity, some special, mature tRNAs have acceptor stems with eight instead of the common seven base pairs. The data from numerous studies suggest that some features in the tRNA domain of pre-tRNAs are important for this behaviour. Here, we show that only five base pairs in the acceptor stem of bacterial histidine tRNAs are required to obtain the changed cleavage site in an unrelated eukaryotic serine tRNA.
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PMID:The acceptor stem in pre-tRNAs determines the cleavage specificity of RNase P. 137 48

Cleavage by RNase P of the tRNA(His precursor yields a mature tRNA with an 8 base pair amino acid acceptor stem instead of the usual 7 base pair stem. Here we show, both in vivo and in vitro, that this is mainly dependent on the primary structure and length of the acceptor stem in the precursor. Furthermore, the tRNA(His) precursor used in this study was processed with a change in both kinetic constants, Km and kcat, in comparison to the kinetics of cleavage of the precursor to tRNA(Tyr)Su3. Cleavage of a chimeric tRNA precursor showed that these altered kinetics were due to a difference in the primary structure and in the length of the acceptor stems of these two tRNA precursors. We also studied the cleavage reaction as a function of base substitutions at positions -1 and/or +73 in the precursor to tRNA(His). Our results suggest that the nucleotide at position +73 in tRNA(His) plays a significant role in the kinetics of cleavage of its precursor, possibly in product release. In addition, it appears that the C5 protein of RNase P is involved in the interaction between the enzyme and its substrate in a substrate-dependent manner, as previously suggested.
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PMID:The kinetics and specificity of cleavage by RNase P is mainly dependent on the structure of the amino acid acceptor stem. 137 49

External guide sequences (EGSs) complementary to mRNAs that encode beta-galactosidase from Escherichia coli and nuclease A from Staphylococcus aureus can target these RNAs for cleavage in vitro by RNase P from E. coli. Specific cleavage occurs at locations predicted by the nucleotide sequences of the EGSs. EGSs with regions complementary to the mRNAs that are as short as 13 nucleotides function efficiently and turn over slowly during incubation with the target substrate and the enzyme. EGSs composed of deoxyribonucleotides as well as those composed of ribonucleotides are effective, but cleavage of the targeted substrate with DNA as an EGS is about 10-fold less efficient than that with RNA as an EGS. An RNA EGS inhibited the formation of beta-galactosidase activity in a crude extract (S30) of E. coli that was capable of catalyzing coupled transcription-translation reactions.
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PMID:Targeted cleavage of mRNA in vitro by RNase P from Escherichia coli. 137 88


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