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)

The rnpA gene from the cyanobacterium Synechocystis sp. PCC 6803, which codes for the protein subunit of ribonuclease P (RNase P), has been cloned by functional complementation of an Escherichia coli mutant. This protein had previously been characterized only in proteobacteria and gram-positive bacteria. rnpA and the closely linked rpmH gene, which code for the large subunit ribosomal protein L34, have been sequenced. The Synechocystis 6803 L34 protein is more similar to the homologous protein from some non-green chloroplasts than to the L34 protein from other bacteria. The protein subunit of RNase P from Synechocystis 6803 has been overexpressed in E. coli and purified to homogeneity. Antibodies raised against the Synechocystis 6803 RNase P protein did not recognize the homologous protein from E. coli (C5 protein). Similarly, antibodies raised against the E. coli C5 protein did not recognize significantly the Synechocystis 6803 protein. In spite of the lack of immunological cross-reactivity and the low level of sequence identity, the E. coli and Synechocystis 6803 proteins are functionally interchangeable. In enzymatic assays using either an E. coli precursor tRNA(Tyr) or a Synechocystis 6803 precursor tRNA(Gln) as substrates, we have detected RNase P activity with holoenzymes reconstituted with the RNA subunit from E. coli and the protein subunit from Synechocystis 6803 or with the RNA subunit from Synechocystis 6803 and the protein subunit from E. coli. The relative efficiency of cleavage of the different substrates is dependent on the origin of the protein subunit used to reconstitute the holoenzyme.
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PMID:Cloning, purification and characterization of the protein subunit of ribonuclease P from the cyanobacterium Synechocystis sp. PCC 6803. 889 83

Ribonuclease P (RNase P) from wheat nuclei has been purified over 1000-fold, using wheat germ extract as starting material and a combination of poly(ethylenglycol) precipitation and column chromatography. The enzyme was shown to be of nuclear origin by its characteristic ionic requirements; for optimum activity it requires 0.5-1.5 mM Mg2+, which can be partly replaced by Mn2+. With about 100 kDa, wheat nuclear RNase P has the lowest molecular mass reported so far for a eukaryotic RNase P. The enzyme has an isoelectric point of 5.0 and a buoyant density of 1.34 g/ml in CsCl, suggesting the presence of a nucleic acid component; it is, however, insensitive against treatment with micrococcal nuclease. Wheat germ RNase P requires an intact tertiary structure of the pre-tRNA substrate; its cleavage efficiency is also influenced by the presence of an intron, and by the nature of the 3' terminus of the substrate. The apparent Km and Vmax for an intronless plant pre-tRNA(Tyr) are 10.3 nM and 1.12 fmol/min, respectively.
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PMID:Partial purification and characterization of nuclear ribonuclease P from wheat. 911 34

Only a few complete sequences and very limited functional data are available for the catalytic RNA component of cyanobacterial RNase P. The RNase P RNA from the chl alb containing cyanobacterium Prochlorothrix hollandica belongs to a rarely found structural subtype with an extended P15/16 domain. We have established conditions for optimal in vitro ribozyme activity, and determined the kinetic parameters for cleavage of pre-tRNA(Tyr). Analysis of pre-tRNA mutants revealed that the T-stem sequence only plays a modulating role, whereas the CCA end is essential for efficient product formation.
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PMID:Sequence and functional characterization of RNase P RNA from the chl alb containing cyanobacterium Prochlorothrix hollandica. 965 27

Eukaryotic transfer RNA precursors (pre-tRNAs) contain a 5' leader preceding the aminoacyl acceptor stem and a 3' trailer extending beyond this stem. An early step in pre-tRNA maturation is removal of the 5' leader by the endoribonuclease, RNase P. Extensive pairing between leader and trailer sequences has previously been demonstrated to block RNase P cleavage, suggesting that the 5' leader and 3' trailer sequences might need to be separated for the substrate to be recognized by the eukaryotic holoenzyme. To address whether the nuclear RNase P holoenzyme recognizes the 5' leader and 3' trailer sequences independently, interactions of RNase P with pre-tRNA(Tyr) containing either the 5' leader, the 3' trailer, or both were examined. Kinetic analysis revealed little effect of the 3' trailer or a long 5' leader on the catalytic rate (k(cat)) for cleavage using the various pre-tRNA derivatives. However, the presence of a 3' trailer that pairs with the 5' leader increases the K(m) of pre-tRNA slightly, in agreement with previous results. Similarly, competition studies demonstrate that removal of a complementary 3' trailer lowers the apparent K(I), consistent with the structure between these two sequences interfering with their interaction with the enzyme. Deletion of both the 5' and 3' extensions to give mature termini resulted in the least effective competitor. Further studies showed that the nuclear holoenzyme, but not the B. subtilis holoenzyme, had a high affinity for single-stranded RNA in the absence of attached tRNA structure. The data suggest that yeast nuclear RNase P contains a minimum of two binding sites involved in substrate recognition, one that interacts with tRNA and one that interacts with the 3' trailer. Furthermore, base pairing between the 5' leader and 3' trailer hinders recognition.
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PMID:Effects of 5' leader and 3' trailer structures on pre-tRNA processing by nuclear RNase P. 1093 10

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.
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PMID:Human tyrosine tRNA is also internally cleavable by E. coli ribonuclease P RNA ribozyme in vitro. 1182 82

Human tyrosine tRNA and fly alanine, histidine, and initiator methionine tRNAs are generally cleavable internally by bacterial ribonuclease P ribozyme. The unusual internal cleavage reaction of tRNA, called hyperprocessing, occurs when the cloverleaf structure of the tRNA molecule is denatured to form a double-hair-pin-like structure. The hyperprocessing reaction of these tRNAs requires magnesium ions. We analyzed details of this reaction using human tyrosine tRNA and Escherichia coli RNase P ribozyme. The usual processing reaction occurred efficiently with magnesium at 5 mM, but for the hyperprpocessing reaction, higher concentrations were needed. With such high concentrations, hyperprocessing cleaved both mature tRNA and tRNA precursor as substrates. When mature tRNA was the substrate, the apparent K(M) was almost the same as in the usual reaction, but k(cat) was smaller. These results indicated that the occurrence of hyperprocessing depends on the magnesium ion concentration, and suggested that magnesium ions contribute to the recognition of the shape of the substrate by bacterial RNase P enzymes.
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PMID:Kinetics of hyperprocessing reaction of human tyrosine tRNA by ribonuclease P ribozyme from Escherichia coli. 1240 Jul 1

Like the translational elongation factor EF-Tu, RNase P interacts with a large number of substrates where RNase P with its RNA subunit generates tRNAs with matured 5' termini by cleaving tRNA precursors immediately 5' of the residue at +1, i.e. at the position that corresponds to the first residue in tRNA. Most tRNAs carry a G+1C+72 base pair at the end of the aminoacyl acceptor-stem whereas in tRNA(Gln) G+1C+72 is replaced with U+1A+72. Here, we investigated RNase P RNA-mediated cleavage as a function of having G+1C+72 versus U+1A+72 in various substrate backgrounds, two full-size tRNA precursors (pre-tRNA(Gln) and pre-tRNA(Tyr)Su3) and a model RNA hairpin substrate (pATSer). Our data showed that replacement of G+1C+72 with U+1A+72 influenced ground state binding, cleavage efficiency under multiple and single turnover conditions in a substrate-dependent manner. Interestingly, we observed differences both in ground state binding and rate of cleavage comparing two full-size tRNA precursors, pre-tRNA(Gln) and pre-tRNA(Tyr)Su3. These findings provide evidence for substrate discrimination in RNase P RNA-mediated cleavage both at the level of binding, as previously observed for EF-Tu, as well as at the catalytic step. In our experiments where we used model substrate derivatives further indicated the importance of the +1/+72 base pair in substrate discrimination by RNase P RNA. Finally, we provide evidence that the structural architecture influences Mg2+ binding, most likely in its vicinity.
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PMID:Substrate discrimination in RNase P RNA-mediated cleavage: importance of the structural environment of the RNase P cleavage site. 1581 65

The 'RNA world' hypothesis holds that during evolution the structural and enzymatic functions initially served by RNA were assumed by proteins, leading to the latter's domination of biological catalysis. This progression can still be seen in modern biology, where ribozymes, such as the ribosome and RNase P, have evolved into protein-dependent RNA catalysts ('RNPzymes'). Similarly, group I introns use RNA-catalysed splicing reactions, but many function as RNPzymes bound to proteins that stabilize their catalytically active RNA structure. One such protein, the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (TyrRS; CYT-18), is bifunctional and both aminoacylates mitochondrial tRNA(Tyr) and promotes the splicing of mitochondrial group I introns. Here we determine a 4.5-A co-crystal structure of the Twort orf142-I2 group I intron ribozyme bound to splicing-active, carboxy-terminally truncated CYT-18. The structure shows that the group I intron binds across the two subunits of the homodimeric protein with a newly evolved RNA-binding surface distinct from that which binds tRNA(Tyr). This RNA binding surface provides an extended scaffold for the phosphodiester backbone of the conserved catalytic core of the intron RNA, allowing the protein to promote the splicing of a wide variety of group I introns. The group I intron-binding surface includes three small insertions and additional structural adaptations relative to non-splicing bacterial TyrRSs, indicating a multistep adaptation for splicing function. The co-crystal structure provides insight into how CYT-18 promotes group I intron splicing, how it evolved to have this function, and how proteins could have incrementally replaced RNA structures during the transition from an RNA world to an RNP world.
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PMID:Structure of a tyrosyl-tRNA synthetase splicing factor bound to a group I intron RNA. 1817 3

The crystal structure of the Alba protein (PhoAlba) from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3, was determined at a resolution of 2.8 A. PhoAlba structurally belongs to the alpha/beta proteins and is similar not only to archaeal homologues but also to RNA-binding proteins, including the C-terminal half of initiation factor 3 (IF3-C) from Bacillus stearothermophilus, an Esherichia coli protein implicated in cell division (Yhhp), and an Arabidopsis protein of unknown function. We found by gel shift assay that PhoAlba interacts with both ribonuclease P (RNase P) RNA (PhopRNA) and precursor-tRNA(Tyr) (pre-tRNA(Tyr)) in P. horikoshii. However, the addition of PhoAlba to reconstituted particles composed of PhopRNA and four or five protein subunits had little influence on either the pre-tRNA processing activity or the optimum temperature for the processing activity. These results suggest that PhoAlba contributes little to the catalytic activity of P. horikoshii RNase P.
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PMID:Crystal structure and functional analysis of an archaeal chromatin protein Alba from the hyperthermophilic archaeon Pyrococcus horikoshii OT3. 1832 60

Modern tools of genomics and proteomics reveal potential therapeutic antisense targets in asthma, increasing the interest in the development of anti-mRNA drugs. In allergic asthma experimental models, antisense oligonucleotides (ASO) are administered by inhalation or systemically. ASO can be used for a large number of molecular targets: cell membrane receptors (G-protein coupled receptors, cytokine and chemokine receptors), membrane proteins, ion channels, cytokines and related factors, signaling non-receptor protein kinases (tyrosine kinases, and serine/threonine kinases) and regulators of transcription belonging to Cys4 zinc finger of nuclear receptor type or beta-scaffold factors with minor groove contacts classes/superclasses of transcription factors. A respirable ASO against the adenosine A(1) receptor was investigated in human trials. RNase P-associated external guide sequence (EGS) delivered into pulmonary tissues represents a potentially new therapeutic approach in asthma as well as ribozyme strategies. Small interfering RNA (siRNA) targeting key molecules involved in the patho-physiology of allergic asthma are expected to be of benefit as RNAi immunotherapy. Antagomirs, synthetic analogs of microRNA (miRNA), have important roles in regulation of gene expression in asthma. RNA interference (RNAi) technologies offer higher efficiency in suppressing the expression of specific genes, compared with traditional antisense approaches.
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PMID:A review of antisense therapeutic interventions for molecular biological targets in asthma. 1970 36


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