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Query: UNIPROT:P06889 (Mol)
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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.
J Mol Biol 1992 Oct 20
PMID:Several regions of a tRNA precursor determine the Escherichia coli RNase P cleavage site. 127 79

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.
J Mol Biol 1992 Jul 20
PMID:Important 2'-hydroxyl groups in model substrates for M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli. 137 4

RNase P is a multi-subunit enzyme responsible for the accurate processing of the 5' terminus of all tRNAs. The RNA subunit from Clostridium sporogenes has been partially purified and characterized. The RNA is approximately 400 nucleotides long and makes a precise endonucleolytic cleavage at the mature 5' terminus of tRNA. The RNA requires moderate concentrations of Mg2+ (20 mM) and relatively high concentrations of NH4Cl (800 mM) for optimal activity. Mn2+ effectively substitutes for Mg2+ at 2 mM. Zn2+, Ni2+, Ca2+, and Co2+ are ineffective at stimulating activity. Monovalent ions are, in general, more effective the greater the ionic radius (NH+4 greater than Cs greater than Rb greater than K greater than Na). In contrast to the activity of Bacillus subtilis, C. sporogenes RNase P RNA is significant more active in (NH4)2SO4 than in NH4Cl.
Mol Microbiol 1990 Aug
PMID:Purification and characterization of RNase P from Clostridium sporogenes. 170 96

We have mapped a gene in the mitochondrial DNA of Candida (Torulopsis) glabrata and shown that it is required for 5' end maturation of mitochondrial tRNAs. It is located between the tRNAfMet and tRNAPro genes, the same tRNA genes that flank the mitochondrial RNase P RNA gene in the yeast Saccharomyces cerevisiae. The gene is extremely AT rich and codes for AU-rich RNAs that display some sequence homology with the mitochondrial RNase P RNA from S. cerevisiae, including two regions of striking sequence homology between the mitochondrial RNAs and the bacterial RNase P RNAs. RNase P activity that is sensitive to micrococcal nuclease has been detected in mitochondrial extracts of C. glabrata. An RNA of 227 nucleotides that is one of the RNAs encoded by the gene that we mapped cofractionated with this mitochondrial RNase P activity on glycerol gradients. The nuclease sensitivity of the activity, the cofractionation of the RNA with activity, and the homology of the RNA with known RNase P RNAs lead us to propose that the 227-nucleotide RNA is the RNA subunit of the C. glabrata mitochondrial RNase P enzyme.
Mol Cell Biol 1991 Mar
PMID:A gene required for RNase P activity in Candida (Torulopsis) glabrata mitochondria codes for a 227-nucleotide RNA with homology to bacterial RNase P RNA. 170 11

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.
J Mol Biol 1991 Feb 20
PMID:Sequence changes in both flanking sequences of a pre-tRNA influence the cleavage specificity of RNase P. 170 37

A study was made of the cleavage by M1 RNA and RNase P of a non-tRNA precursor that can serve as a substrate for RNase P from Escherichia coli, namely, the precursor to 4.5 S RNA (p4.5S). The overall efficiency of cleavage of p4.5S by RNase P is similar to that of wild-type tRNA precursors. However, unlike the reaction with wild-type tRNA precursors, the reaction catalyzed by the holoenzyme with p4.5S as substrate has a much lower Km value than that catalyzed by M1 RNA with the same substrate, indicating that the protein subunit plays a crucial role in the recognition of p4.5S. A model hairpin substrate, based on the sequence of p4.5S, is cleaved with greater efficiency than the parent molecule. The 3'-terminal CCC sequence of p4.5 S may be as important for cleavage of this substrate as the 3'-terminal CCA sequence is for cleavage of tRNA precursors.
J Mol Biol 1991 Sep 05
PMID:Kinetics of the processing of the precursor to 4.5 S RNA, a naturally occurring substrate for RNase P from Escherichia coli. 171 93

A 40-kDa To antigen recognized by sera from some patients with autoimmune diseases is an integral component of both human RNase P and mitochondrial RNA processing (MRP) RNase. Human MRP and RNase P RNAs, synthesized in vitro, readily associate with the To antigen present in the HeLa cell extract. Using this in vitro reconstitution system, the binding site of the To antigen is localized to a 44-nucleotide-long sequence corresponding to nucleotides 21 to 64 of the human MRP RNA. UV cross-linking experiments showed that the To antigen binds directly to MRP RNA and to RNase P (H1) RNA through RNA-protein interactions. Although the MRP RNA and RNAse P (H1) RNA show sequence homology in four conserved blocks (H. A. Gold, J. N. Topper, D. A. Clayton, and J. Craft, Science 245:1377-1380, 1989), the To antigen-binding site in MRP RNA does not show any obvious primary sequence homology with H1 RNA. These data suggest that the To antigen binds to a conserved and presumably a common secondary or tertiary structure in human MRP and RNase P RNAs.
Mol Cell Biol 1991 Oct
PMID:The 40-kilodalton to autoantigen associates with nucleotides 21 to 64 of human mitochondrial RNA processing/7-2 RNA in vitro. 171 26

Cells overexpressing the RNA-processing enzymes RNase III, RNase E and RNase P were fractionated into membrane and cytoplasm. The RNA-processing enzymes were associated with the membrane fraction. The membrane was further separated to inner and outer membrane and the three RNA-processing enzymes were found in the inner membrane fraction. By assaying for these enzymatic activities we showed that even in a normal wild-type strain of Escherichia coli these enzymes fractionate primarily with the membrane. The RNA part of RNase P is found in the cytosolic fraction of cells overexpressing this RNA, while the overexpressed RNase P protein sediments with the membrane fraction; this suggests that the RNase P protein anchors the RNA catalytic moiety of the enzyme to a larger entity. The implications of these findings for the cellular organization of the RNA-processing enzymes in the cell are discussed.
Mol Microbiol 1991 Jul
PMID:Location of the RNA-processing enzymes RNase III, RNase E and RNase P in the Escherichia coli cell. 194 11

RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein. We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.
Mol Cell Biol 1991 Feb
PMID:Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P. 199 Feb 78

One addition mutation and several small deletion mutations have been created in vitro at a unique site in the gene coding for M1 RNA, the RNA subunit of Escherichia coli RNase P. The mutant genes exhibit a wide range of efficiencies in complementing another mutant that is thermosensitive for RNase P function in vivo. The transcripts of the mutated genes cleave a precursor tRNA in vitro with efficiencies that parallel their ability to function in the complementation assay in vivo. The secondary structures in solution of the mutant gene transcripts are shown to be different from the parent molecule by probing the structure of the transcripts with ribonuclease T1. A local region of secondary structure, between nucleotides 275 and 295, must be maintained for normal function of M1 RNA.
J Mol Biol 1986 Sep 20
PMID:Site-directed mutagenesis of M1 RNA, the RNA subunit of Escherichia coli ribonuclease P. The effects of an addition and small deletions on catalytic function. 243 55


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