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

---

Query: EC:3.1.26.3 (RNase III)
1,015 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The small protein (VPg) covalently linked to the 5' end of poliovirus Type 1 (PV-1) RNA has been labeled in vitro with 125I using the Bolton and Hunter reagent. The RNA is not degraded under the conditions used and nearly all the label enters VPg and not the poly-nucleotide chain. When this 125I-labeled RNA is cleaved with RNase III at low monovalent salt concentrations, one major 125I-labeled fragment, approximately 100 nucleotides long, is produced. The corresponding fragment from similar digests of 32P-labeled RNA has also been identified. The 32P-labeled fragment changes electrophoretic mobility after protease treatment indicating that it contains VPg. Furthermore, the RNase T1 oligonucleotide known to be at the 5' terminus of poliovirus RNA is found in T1 digests of the purified fragment. These results confirm that the fragment is derived from the 5' end of the RNA. This fragment will be useful in studies concerning the initiation of protein synthesis during poliovirus infection.
...
PMID:Identification of specific fragments containing the 5' end of poliovirus RNA after ribonuclease III digestion. 21 65

The procaryotic RNA processing enzyme RNase III (endoribonuclease III [EC 3.1.4.24]) was used to probe vesicular stomatitis virus (VSV) RNAs for specific sites that could be recognized and cleaved. The effect of the enzyme on the RNAs was monitored by measuring their subsequent migration in denaturing agarose-urea gels. VSV virion RNA (negative strand; Mr, 4 X 10(6)) was cleaved by the enzyme to yield a set of discrete fragments which ranged on size from 3.5 X 10(6) to 0.2 X 10(6) daltons. The cleavage was a function of enzyme concentration, salt concentration, and time. A maximum of 20 to 22 fragments was generated under conditions of low enzyme concentration or short times of incubation. VSV genome-length intracellular RNA of both + and - polarity was also cleaved by RNase III. In contrast to the findings with virion-length RNA, however, the migration rates of VSV mRNA's purified by chromatography on polyuridylic acid-Sepharose were unaffected by treatment with RNase III. These results show that specific sites in the virion RNA and its full-length complement can be recognized by RNase III. Sites of this type are not present in the polyadenylic acid-containing mRNA, however.
...
PMID:RNase III cleaves vesicular stomatitis virus genome-length RNAs but fails to cleave viral mRNA's. 22 9

Heterogeneous nuclear RNA (hnRNA) from HeLa cells contains intramolecular duplexes. Since hnRNA is associated with protein in vivo, it is possible that the double-stranded regions observed in deproteinized hnRNA form spontaneously upon the release of protein from single-stranded but potentially complementary sequences. We show here that this is not the case for a class of double-stranded sequences that is defined by resistance to RNases A + T(1) at high ionic strength. Exposure of HeLa hnRNA.ribonucleoprotein (hnRNP) particles to Escherichia coli RNase III, a double-strand-specific endoribonuclease, destroys most of the sequences resistant to RNases A + T(1). This effect is completely blocked when hnRNP is exposed to RNase III in the presence of an excess of purified double-stranded RNA. In addition, we show that there exist two classes of double-stranded RNA in hnRNP at a salt concentration of 0.13 M. These are distinguished by their relative resistance to RNases A + T(1). The more stable double-stranded sequences, which are resistant to RNases A + T(1) at 0.13 M, comprise 1.0-1.1% of the nucleotides in hnRNP. The less stable double-stranded sequences comprise an additional 1.5-2.0% of the nucleotides in hnRNP. These are sensitive to RNase III at 0.13 M, but are not resistant to RNases A + T(1) unless the salt concentration is raised to 0.63 M. The demonstration that double-stranded sequences resistant to RNases A + T(1) exist in native ribonucleoprotein and are not artifacts of deproteinization now makes it appropriate to seriously consider their possible functional role in hnRNA metabolism, perhaps as binding sites for regulatory proteins involved in mRNA processing.
...
PMID:Secondary structure of heterogeneous nuclear RNA: two classes of double-stranded RNA in native ribonucleoprotein. 41 25

Nucleoli of both chick embryos and mouse Ehrlich ascites cells contain an enzymatic activity that is very similar to RNase DII, an enzyme isolated from total chick embryos for its ability to degrade double-stranded RNA. The enzyme can be extracted by low salt/EDTA from nucleoli and is associated with pre-ribosomal 80-S and 55-S particles. Under ionic conditions which are inhibitory for the nucleolytic activity the transcript in vitro of nucleoli is not processed and sediments around 45 S. Under salt conditions which are optimal for the nucleolar enzyme the nucleolar transcripts are cleaved to distinct intermediate-sized molecules. Addition of the chicken RNase DII or RNase III to the nucleolar transcription system results in a similar shift of the chain length of the RNA molecules. It is concluded that a nucleolar RNase recognizing double-stranded regions in the pre-ribosomal RNA is involved in the maturation of ribosomal RNA.
...
PMID:Localisation of an endonuclease specific for double-stranded RNA within the nucleolus and its implication in processing ribosomal transcripts. 42 96

T4 Species I RNA, a molecule 140 nucleotides in length with some structural features very much like a tRNA, is specifically cleaved by an enzymatic activity in Escherichia coli extracts to give three segments with 19, 48 and 73 nucleotides. We report the purification and characterization of the E. coli RNase which cleaves two 3' phosphodiester bonds of T4 Species I RNA. This reaction has many properties in common with those catalyzed by E. coli RNase III, although the optimal salt conditions for T4 Species I RNA cleavage differ significantly from those for other RNase III-catalyzed reactions. The reaction is not catalyzed by extracts from an E. coli strain lacking RNase III activity. Furthermore, T4 Species I RNA is cleaved by highly purified E. coli RNase III to yield the same three specific fragments. We conclude that this specific cleavage is due to the action of RNase III, and that the requirement for lower ionic strength may reveal further important properties about this RNA processing enzyme.
...
PMID:Cleavage of T4 species I ribonucleic acid by Escherichia coli ribonuclease III. 78 26

Double-stranded RNAs from Penicillium chrysogenum virus have been treated with RNAse III, pancreatic RNAse A and RNAse T1 and the degradation of the RNAs has been studied under different conditions. It was found that only the two former enzymes cut across both strands, RNase T1 cannot cleave double strands. RNase III was shown to digest double-stranded RNA by a two step process: an initial phase of specific cleavage is followed by random degradation. In the first phase the enzyme exhibited a definite preference for some specific base pattern. Partial or complete degradation with pancreatic RNase A could also be achieved in media with high salt concentration provided that the enzyme: substrate ratio was increased together with the salt concentration. By combining different assay techniques, the process of degradation was followed from the early stages to complete digestion and the breakdown products were characterised. It is suggested that a structural change in the enzyme molecules enables them to act on double-stranded RNA. RNAse T1, being unable to cleave double strands, provides a useful tool for studying the secondary structure of RNA molecules. Treatment with different nucleases yielded some new information on the structure of different RNA species in Penicillium stoloniferum virus.
...
PMID:Action of nucleases on double-stranded RNA. 81 98

An Escherichia coli double strand specific endoribonuclease, RNase III, was cloned, expressed in large amounts, and purified to homogeneity. Enzyme activity was monitored by assaying fractions for the ability to correctly process exogenous RNA containing specific RNase III cleavage sites. DEAE-Sepharose ion exchange chromatography in the presence of a linear KCl gradient (from 0.02 M to 0.75 M) demonstrated that RNase III exists as two distinct forms. One form elutes at a KCl concentration of 0.13 M and the other elutes at 0.33 M. The presence of stoichiometric amounts of the GTP-binding protein Era during purification results in the conversion of the low salt form into the high salt form. Size exclusion chromatography demonstrated that both forms exist as a dimer in solution. In order to investigate the nature of the dimer, protein cross-linking was performed and cross-linked products were detected by silver staining. The protein-protein dimer can be visualized at protein:cross-linker molar ratios as low as 1:15 within 1 minute of exposure to cross-linker in 0.1 M KCl. Upon addition of substrate RNA to the cross-linking reaction a second form of the protein-protein dimer (with a slightly smaller apparent molecular weight) becomes prominent. Induction of the new form is absolutely dependent upon the addition of substrate mRNA to the reaction mixture. We postulate that the RNase III dimer undergoes a dramatic conformational change upon recognition of RNA which we are able to trap by cross-linking.
...
PMID:Characterization of the biochemical properties of recombinant ribonuclease III. 169 24

The structure of a ribonuclease III processing signal from bacteriophage T7 was examined by NMR spectroscopy, optical melting, and chemical and enzymatic modification. A 41 nucleotide variant of the T7 R1.1 processing signal has two Watson-Crick base-paired helices separated by an internal loop, consistent with its predicted secondary structure. The internal loop is neither rigidly structured nor completely exposed to solvent, and seems to be helical. The secondary structure of R1.1 RNA is largely insensitive to the monovalent cation concentration, which suggests that the monovalent cation sensitivity of secondary site cleavage by RNase III is not due to a low salt-induced RNA conformational change. However, spectroscopic data show that Mg2+ affects the conformation of the internal loop, suggesting a divalent cation binding site(s) within this region. The Mg(2+)-dependence of RNase III processing of some substrates may reflect not only a requirement for a divalent cation as a catalytic cofactor, but also a requirement for a local RNA conformation which is divalent cation-stabilized.
...
PMID:Structural characterization of a ribonuclease III processing signal. 812 10

A mutational approach was employed to identify sequence and structural elements in a ribonuclease III processing signal that are important for in vitro enzymatic cleavage reactivity and selectivity. The substrate analyzed was the bacteriophage T7 R1.1 processing signal, a 60 nucleotide irregular RNA hairpin exhibiting an upper and lower dsRNA stem, separated by an asymmetric internal loop which contains the scissile phosphodiester bond. Altering the length of either the upper or lower dsRNA segment in R1.1 RNA dose not change the site of RNase III cleavage. However, decreasing the size of either the upper or lower dsRNA segment causes a progressive inhibition of processing reactivity. Omitting monovalent salt from the reaction buffer promotes cleavage of otherwise unreactive R1.1 deletion mutants. Accurate processing is maintained with R1.1 variants containing specific point mutations, designed to disrupt Watson-Crick (WC) base-pairing in a conserved sequence element within the upper dsRNA stem. The internal loop is not required for processing reactivity, as RNase III can accurately and efficiently cleave R1.1 variants in which this structure is WC base-paired. Moreover, an additional cleavage site is utilized in these variants, which occurs opposite the canonical site, and is offset by two nucleotides. The fully base-paired R1.1 variants form a stable complex with RNase III in Mg(2+)-free buffer, which can be detected by a gel electrophoretic mobility shift assay. In contrast, the complex of wild-type R1.1 RNA with RNase III is unstable during nondenaturing gel electrophoresis. Thus, a functional role of the T7 R1.1 internal loop is to enforce single enzymatic cleavage, which occurs at the expense of RNase III binding affinity.
...
PMID:Mutational analysis of a ribonuclease III processing signal. 833 52

Escherichia coli ribonuclease III, purified to homogeneity from an overexpressing bacterial strain, exhibits a high catalytic efficiency and thermostable processing activity in vitro. The RNase III-catalyzed cleavage of a 47 nucleotide substrate (R1.1 RNA), based on the bacteriophage T7 R1.1 processing signal, follows substrate saturation kinetics, with a Km of 0.26 microM, and kcat of 7.7 min.-1 (37 degrees C, in buffer containing 250 mM potassium glutamate and 10 mM MgCl2). Mn2+ and Co2+ can support the enzymatic cleavage of the R1.1 RNA canonical site, and both metal ions exhibit concentration dependences similar to that of Mg2+. Mn2+ and Co2+ in addition promote enzymatic cleavage of a secondary site in R1.1 RNA, which is proposed to result from the altered hydrolytic activity of the metalloenzyme (RNase III 'star' activity), exhibiting a broadened cleavage specificity. Neither Ca2+ nor Zn2+ support RNase III processing, and Zn2+ moreover inhibits the Mg(2+)-dependent enzymatic reaction without blocking substrate binding. RNase III does not require monovalent salt for processing activity; however, the in vitro reactivity pattern is influenced by the monovalent salt concentration, as well as type of anion. First, R1.1 RNA secondary site cleavage increases as the salt concentration is lowered, perhaps reflecting enhanced enzyme binding to substrate. Second, the substitution of glutamate anion for chloride anion extends the salt concentration range within which efficient processing occurs. Third, fluoride anion inhibits RNase III-catalyzed cleavage, by a mechanism which does not involve inhibition of substrate binding.
...
PMID:Ribonuclease III cleavage of a bacteriophage T7 processing signal. Divalent cation specificity, and specific anion effects. 849 5


1 2 3 Next >>

---