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:3.1.27.3 (RNase T1)
1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In addition to the RI (replicative intermediate RNA) and native RF (replicative form RNA), mouse hepatitis virus-infected cells contained six species of RNA intermediates active in transcribing subgenomic mRNA. We have named these transcriptive intermediates (TIs) and native transcriptive forms (TFs) because they are not replicating genome-sized RNA. Based on solubility in high salt solutions, approximately 70% of the replicating and transcribing structures that accumulated in infected cells by 5-6 h post-infection were multi-stranded intermediates, the RI/TIs. The other 30% were in double-stranded structures, the native RF/TFs. These replicating and transcribing structures were separated by velocity sedimentation on sucrose gradients or by gel filtration chromatography on Sepharose 2B and Sephacryl S-1000, and migrated on agarose gels during electrophoresis, according to their size. Digestion with RNase T1 at 1-10 units/microgram RNA resolved RI/TIs into RF/TF cores and left native RF/TFs intact, whereas RNase A at concentrations of 0.02 microgram/microgram RNA or higher degraded both native RF/TFs and RI/TIs. Viral RI/TIs and native RF/TFs bound to magnetic beads containing oligo(dT)(25), suggesting that the poly(A) sequence on the 3' end of the positive strands was longer than any poly(U) on the negative strands. Kinetics of incorporation of [(3)H]uridine showed that both the RI and TIs were transcriptionally active and the labelling of RI/TIs was not the dead-end product of aberrant negative-strand synthesis. Failure originally to find TIs and TF cores was probably due to overdigestion with RNase A.
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PMID:The RNA structures engaged in replication and transcription of the A59 strain of mouse hepatitis virus. 1116 Dec 78

RNase I and RNase T1 can be used to obtain high-quality footprinting information for paromomycin binding to a 176-mer RNA from the packaging region of HIV-1 (LAI). Controls and scanning procedures are necessary for quantitation of autoradiographic data, so that footprinting plots showing cutting behavior as a function of drug concentration can be used to identify binding sites and regions of altered structure on the 176-mer. From the RNase I footprinting results the primary paromomycin binding sites on the 176-mer are on the main stem and on the stem of SL1, but noncontiguous sequences may be involved in the same binding event. Strong enhancements in cleavage with added drug are also observed, indicating drug-induced structural changes. Drug binding may cause linker regions between stem-loops of the 176-mer to change structure, possibly providing a site or sites for additional drug binding. Because drug binding changes the structure of the packaging region, which may alter its function, paromomycin analogs with enhanced specificity for HIV psi RNA have potential as a new class of agent for treating AIDS.
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PMID:Drug-RNA footprinting. 1149 62

Mutation detection based on ribonuclease cleavage of basepair mismatches in single-stranded RNA probes hybridized to DNA targets was first described over 15 years ago. The original methods relied on RNase A for mismatch cleavage; however, this enzyme fails to cleave many mismatches and has other drawbacks. More recently, a new method for RNase-cleavage-based mutation scanning has been developed, which takes advantage of the ability of RNase 1 and RNase T1 to cleave mismatches in duplex RNA targets, when these enzymes are used in conjunction with nucleic acid intercalating dyes. The method, called NIRCA, is relatively low-cost in terms of materials and equipment required. It is being used to detect mutations and SNPs in a wide variety of genes involved in human genetic disease and cancer, as well as in disease-related viral and bacterial genes. This review describes historical and recently developed RNase cleavage-based methods for mutation/SNP scanning.
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PMID:RNase cleavage-based methods for mutation/SNP detection, past and present. 1152 30

Ribonucleases are ubiquitous in distribution. Ribonucleases that hydrolyse RNA to 3' mononucleotides via 2', 3' cyclic nucleotides are classified into three groups, RNase A, RNase T1, and RNase T2 families. Apart from salvage of cellular or extracellular RNAs, RNases participate in vital cellular functions such as DNA replication, transcription and RNA processing, splicing and editing, and control of translation by determining the turnover of RNA. T2 family RNases have been implicated in nutrition, phosphate remobilization, self-incompatibility, senescence, and defense against pathogens. They are important analytical enzymes and have been exploited for the structural determination of RNAs. Although considerable information is available on RNase A and T1 family RNases, less information is available on RNases from T2 family except RNase Rh from Rhizopus niveus and RNase LE from tomato. However, during the last few years, the primary structure, active site nature based on sequence homology, and probable mechanism of action have been postulated for some of these enzymes. RNases of T2 family, their occurrence, purification, characteristics, biological role, and applications have been reviewed.
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PMID:Ribonucleases from T2 family. 1210 72

We have studied the interaction of the aminoglycoside drug, paromomycin, with a 171-mer from the packaging region of HIV-1 (psi-RNA), using quantitative footprinting and circular dichroism spectroscopy. The footprinting autoradiographic data were obtained by cutting end-labeled RNA with RNase I or RNase T1 in the presence of varying paromomycin concentrations. Scanning the autoradiograms produced footprinting plots showing cleavage intensities for specific sites on the psi-RNA as functions of drug concentration. Footprinting plots showing binding were analyzed using a two-state model to give apparent binding constants for specific sites of the psi-RNA. These plots show that the highest-affinity paromomycin binding site involves nucleotides near bulges in the main stem and SL-1, and other nucleotides in SL-4 of the psi-RNA. RNase I gives an apparent value of K for this drug site of approximately 1.7 x 10(5) M(-1) while RNase T1 reports a value of K of approximately 8 x 10(4) M(-1) (10 mM Tris HCl, pH 7). Footprinting shows that loading the highest affinity site with paromomycin causes structural changes in the single-stranded linker regions, between the stem-loops and main stem and the loops of SL-1 and SL-3. Drug-induced structural changes also affect the intensity of the 208 nm band in the circular dichroism spectrum of the psi-RNA. Fitting the changes in CD band intensity to a two-state model yielded a binding constant for the highest-affinity drug site of 6 x 10(6) M(-1). Thus, the binding constants from footprinting are lower than those obtained for the highest-affinity site from the circular dichroism spectrum, and lower than those earlier obtained using absorption spectroscopy (Sullivan, J. M.; Goodisman, J.; Dabrowiak, J. C., Bioorg. Med. Chem. Lett. 2002, 12, 615). The discrepancy may be due to competitive binding between drug and cleavage agent in the footprinting experiments, but other explanations are discussed. In addition to revealing sites of binding and regions of drug-induced structural change, footprinting showed that the loop regions of SL-1, SL-3 and SL-4 are exposed in the RNA, whereas the linker region between SL-1 and SL-2 is 'buried' and not accessible to cutting by RNase I or RNase T1.
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PMID:Footprinting and circular dichroism studies on paromomycin binding to the packaging region of human immunodeficiency virus type-1. 1221 82

Mass spectrometry plays a central role in the characterisation of modified nucleotides, but pseudouridine is a mass-silent post-transcriptional modification and hence not detectable by direct mass spectrometric analysis. We show by the use of matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry that pseudouridines in tRNA can be specifically cyanoethylated by acrylonitrile without affecting the uridines. The tRNA was cyanoethylated and then subjected to digestion with either RNase A or RNase T1. Cyanoethylated digestion fragments were identified by mass spectrometric comparison of untreated and acrylonitrile-treated samples, where the addition of one acrylonitrile resulted in a mass increment of 53.0 Da. The exact modified nucleotide could be identified by tandem mass spectrometry on the cyanoethylated digestion fragment. The methodology was used to identify additional one 4-thiouridine and one pseudouridine in tRNA(TyrII) from Escherichia coli. Furthermore, we observed that RNase A is highly tolerant towards nucleotide modifications, only being inhibited by 2'-O-methylation, whereas RNase T1 cleavage is affected by most nucleotide modifications.
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PMID:Detection of pseudouridine and other modifications in tRNA by cyanoethylation and MALDI mass spectrometry. 1246 67

By reason of their cytotoxicity, ribonucleases (RNases) are potential anti-tumor drugs. Particularly members from the RNase A and RNase T1 superfamilies have shown promising results. Among these enzymes, Onconase, an RNase from the Northern Leopard frog, is furthest along in clinical trials. A general model for the mechanism of the cytotoxic action of RNases includes the interaction of the enzyme with the cellular membrane, internalization, translocation to the cytosol, and degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the capability of the RNase to evade the intracellular RNase inhibitor has not yet been fully elucidated. This paper discusses the various approaches to exploit RNases as cytotoxic agents.
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PMID:Natural and engineered ribonucleases as potential cancer therapeutics. 1690 46

Replication of the ColE2 plasmid requires a plasmid-coded initiator protein (Rep). Rep expression is controlled by antisense RNA (RNAI) against the Rep mRNA at a translational step. In this paper, we examined the effects of host RNA degradation enzymes on the degradation process of the Rep mRNA and its degradation intermediates especially those carrying the 5' untranslated region. We showed that the Rep mRNA is subjected to complex degradation pathways involving at least RNase I, RNase II, RNase III, RNase E, RNase G and PNPase. RNase II acts as a major exoribonuclease and PNPase plays a minor role. We also showed that the PcnB (polyA polymerase I) plays only a minor role in the Rep mRNA degradation process. The RNA degradation pathways of the Rep mRNA and RNAI of the ColE2 plasmid are quite different. Based on these results, we speculate that the ColE2 Rep mRNA and RNAI are endowed with individual RNA half lives required for the efficient copy number control by being subjected to different RNA degradation systems.
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PMID:Replication initiator protein mRNA of ColE2 plasmid and its antisense regulator RNA are under the control of different degradation pathways. 1819 Dec 5

Ribonucleases (RNases) with different sequence specificities are used for a variety of analytical purposes, including RNA sequencing, mapping, and quantitation. One very common application for RNase A is presented in this unit and involves hydrolyzing RNA that contaminates DNA preparations. Two other commonly used RNases, RNase H and RNase T1, are also described. In addition, many commercially available RNases are sequence-specific endoribonucleases, a property has been used for enzymatic sequencing of RNA.
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PMID:Ribonucleases. 1826 22

Individual transfer ribonucleic acids (tRNAs) in a complex mixture can be identified by the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) detection of their signature digestion products. Signature digestion products are endonuclease digestion products whose mass-to-charge value is unique thus corresponding to only a single tRNA. To improve the effectiveness of this approach, we have expanded the applicable endonucleases and examined the use of multiple endonucleases for tRNA identification. The purine specific endonucleases RNase T1 and RNase TA generate the largest number of predicted signature digestion products. Experimentally, MALDI-MS analysis of endonuclease digests from Escherichia coli and Bacillus subtilis finds that any two endonucleases used in combination increases tRNA identification by about 25% over the number identified with a single endonuclease. Using three endonucleases, RNase T1, RNase A, and RNase TA, further improves the number of tRNAs identified by 10-15% over those found with two endonucleases. Limitations in the MALDI-MS approach for complex mixtures were revealed in this study, suggesting that the direct MALDI-MS analysis of signature digestion products is more effective for organisms having 30 or less unique tRNAs.
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PMID:Multiple endonucleases improve MALDI-MS signature digestion product detection of bacterial transfer RNAs. 1910 81


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