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

A RNase from calf thymus, which specifically cleaves native or synthetic double-stranded RNA molecules endonucleolytically, has been isolated and purified from calf thymus. For optimal activity, the enzyme requires a sulfhydryl reagent and divalent cations; over 95 per cent of the activity is inhibited by 0.5 mm ethidium bromide. The degradation of [3H]poly(C)-poly(I) by purified enzyme preparations yields labeled dinucleotides and octanucleotides; the latter oligonucleotide contained 5'-phosphate and 3'-hydroxyl termini. The enzyme cleaves high molecular weight RNAs such as RNA products formed in vitro by T3 phage-induced RNA polymerase from T3 phage DNA, heterogeneous RNA isolated from duck reticulocyte nuclei, and 45 S RNA isolated from rat liver nucleoli. The mode of degradation of RNA in vitro with the double-stranded RNase is similar to that of Escherichia coli RNase III and appears to act endonucleolytically. The degradation of 45 S RNA with the enzyme results in the production of 29 S and 19 S RNA fragments. These findings suggest that the enzyme may be involved in the processing of high molecular weight precursor RNAs to mRNA or rRNAs in a manner analogous to that reported for RNase III of E. coli.
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PMID:Isolation and purification of double-stranded ribonuclease from calf thymus. 83 40

We have studied the nuclease activities present in preparations of Escherichia coli RNase III and the "sizing factor" responsible for specific processing of several RNA species. RNase III preparations contain three activities: one which solubilizes stable RNA:RNA duplexes; one which solubilizes the RNA of DNA:RNA hybrids; and one which processes the polycistronic mRNA of bacteriophage T7 in a manner identical with sizing factor. We show that the activity against the RNA of DNA:RNA hybrids can be removed, but that the activity which cleaves RNA:RNA duplexes and that responsible for specific processing of phage T7 polycistronic mRNA appear to be identical by several biochemical criteria. In addition, partially purified enzyme fractions from mutants lacking these two activities contain substantial amounts of activity against the RNA of DNA:RNA hybrids. We have also defined several properties of the two activities solubilize RNA:RNA duplexes and RNA of DNA:RNA hybrids. Average oligonucleotide chain length in an exhaustive digest of double-stranded RNA is about 15 bases, while that in a digest of the RNA in DNA:RNA hybrids is less than 10 bases. Direct analysis shows that both activities cleave RNA chains to yield 5'-phosphate and 3'-hydroxyl termini. All four bases can reside at the 5' end of the resulting oligonucleotides, although both activities show a mild preference for certain bases. These results and previous findings allow us to specify the probably size and structure of potential cleavage sites for these enzymes in biological RNA molecules.
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PMID:Ribonucleic acid processing activity of Escherichia coli ribonuclease III. 109 44

the mutation that causes ribonuclease III (RNase III) deficiency in strain AB301-105 of Kindler et al. (1973) has been mapped by use of F' merodiploids, Hfr matings, and P1 transduction. This mutation, rnc-105, lies close to nadB, near 49 min on the genetic map of Escherichia coli. The rnc-105 mutation has been transferred from its original genetic background by transduction and conjugation, and these new strains have the same defects in ribonucleic acid processing reported previously for AB301-105. Strains that carry rnc-105 grow more slowly than parental rnc+ strains, but the difference in growth rate seems to depend on the genetic background of each strain. Bacteriophage T7 grows about equally well in RNase III+ and III- female strains of E. coli, even though the specific cuts that RNase III makes in T7 ribonucleic acid are not made in the RNase III- strains. A low-phosphate defined medium in which most E. coli strains seem to grow well was developed. This medium is equally useful for labeling ribonucleic acids with 32PO4 and as a selective medium for genetic manipulations. It was used to determine the growth requirements of strain AB301-105, which are biotin and succinate in addition to the methionine and histidine requirements of the parental strain. The biotin mutation lies near the position expected from known mutations of E. coli, but the succinate mutation apparently does not. The possibility that the succinate requirement could be due to the RNase III deficiency is discussed. A uraP mutation was isolated for use in transferring rnc-105 between strains by conjugation. It lies near 47 min, somewhat removed from the commonly accepted position for uraP.
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PMID:Genetic mapping of a mutation that causes ribonucleases III deficiency in Escherichia coli. 110 Jun 5

RNase III activity of Escherichia coli is stimulated 4-fold after infection with bacterial virus T7. The mechanism of stimulation is based on phosphate transfer to RNase III by the T7 coded protein kinase. In vitro synthesized protein kinase also stimulates RNase III. Serine is the phosphate acceptor.
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PMID:RNase III is positively regulated by T7 protein kinase. 640 99

Bacteriophage T7 expresses a serine/threonine-specific protein kinase activity during infection of its host, Escherichia coli. The protein kinase (gp0.7 PK), encoded by the T7 early gene 0.7, enhances phage reproduction under sub-optimal growth conditions. It was previously shown that ribosomal protein S1 and translation initiation factors IF1, IF2, and IF3 are phosphorylated in T7-infected cells, and it was suggested that phosphorylation of these proteins may serve to stimulate translation of the phage late mRNAs. Using high-resolution two-dimensional gel electrophoresis and specific immunoprecipitation, we show that elongation factor G and ribosomal protein S6 are phosphorylated following T7 infection. The gel electrophoretic data moreover indicate that elongation factor P is phosphorylated in T7-infected cells. T7 early and late mRNAs are processed by ribonuclease III, whose activity is stimulated through phosphorylation by gp0.7 PK. Specific overexpression and phosphorylation was used to locate the RNase III polypeptide in the standard two-dimensional gel pattern, and to confirm that serine is the phosphate-accepting amino acid. The two-dimensional gels show that the in vivo expression of gp0.7 PK results in the phosphorylation of over 90 proteins, which is a significantly higher number than previous estimates. The protein kinase activities of the T7-related phages T3 and BA14 produce essentially the same pattern of phosphorylated proteins as that of T7. Finally, several experimental variables are analysed which influence the production and pattern of phosphorylated proteins in both uninfected and T7-infected cells.
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PMID:Phosphorylation of elongation factor G and ribosomal protein S6 in bacteriophage T7-infected Escherichia coli. 802 76

Ethylation interference and hydroxyl radical footprinting were used to identify substrate ribose-phosphate backbone sites that interact with the Escherichia coli RNA processing enzyme, ribonuclease III. Two RNase III mutants were employed, which bind substrate in vitro similarly as wild-type enzyme, but lack detectable phosphodiesterase activity. Specifically, altering glutamic acid at position 117 to lysine or alanine uncouples substrate binding from cleavage. The two substrates examined are based on the bacteriophage T7 R1.1 RNase III processing signal. One substrate, R1.1 RNA, undergoes accurate single cleavage at the canonical site, while a close variant, R1.1[WC-L] RNA, undergoes coordinate double cleavage. The interference and footprinting patterns for each substrate (i) overlap, (ii) exhibit symmetry and (iii) extend approximately one helical turn in each direction from the RNase III cleavage sites. Divalent metal ions (Mg2+, Ca2+) significantly enhance substrate binding, and confer stronger protection from hydroxyl radicals, but do not significantly affect the interference pattern. The footprinting and interference patterns indicate that (i) RNase III contacts the sugar-phosphate backbone; (ii) the RNase III-substrate interaction spans two turns of the A-form helix; and (iii) divalent metal ion does not play an essential role in binding specificity. These results rationalize the conserved two-turn helix motif seen in most RNase III processing signals, and which is necessary for optimal processing reactivity. In addition, the specific differences in the footprint and interference patterns of the two substrates suggest why RNase III catalyzes the coordinate double cleavage of R1.1[WC-L] RNA, and dsRNA in general, while catalyzing only single cleavage of R1.1 RNA and related substrates in which the scissle bond is within an asymmetric internal loop.
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PMID:Defining the enzyme binding domain of a ribonuclease III processing signal. Ethylation interference and hydroxyl radical footprinting using catalytically inactive RNase III mutants. 863 75

The pac1+ gene of the fission yeast Schizosaccharomyces pombe is essential for viability and its overexpression induces sterility and suppresses mutations in the pat1+ and snm1+ genes. The pac1+ gene encodes a protein that is structurally similar to RNase III from Escherichia coli, but its normal function is unknown. We report here the purification and characterization of the Pac1 protein after overexpression in E. coli. The purified protein is a highly active, double-strand-specific endoribonuclease that converts long double-stranded RNAs into short oligonucleotides and also cleaves a small hairpin RNA substrate. The Pac1 RNase is inhibited by a variety of double- and single-stranded polynucleotides, but polycytidylic acid greatly enhances activity and also promotes cleavage specificity. The Pac1 RNase produces 5'-phosphate termini and requires Mg2+; Mn2+ supports activity but causes a loss of cleavage specificity. Optimal activity was obtained at pH 8.5, at low ionic strength, in the presence of a reducing agent. The enzyme is relatively insensitive to N-ethylmaleimide but is strongly inhibited by ethidium bromide and vanadyl ribonucleoside complexes. The properties of the Pac1 RNase support the hypothesis that it is a eukaryotic homolog of RNase III.
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PMID:Purification and characterization of the Pac1 ribonuclease of Schizosaccharomyces pombe. 871 May 10

A 320-nucleotide RNA with several characteristic features was expressed in Bacillus subtilis to study RNA processing. The RNA consisted of a 5'-proximal sequence from bacteriophage SP82 containing strong secondary structure, a Bs-RNase III cleavage site, and the 3'-proximal end of the ermC transcriptional unit. Comparison of RNA processing in a wild-type strain and a strain in which the pnpA gene, coding for polynucleotide phosphorylase (PNPase), was deleted, as well as in vitro assays of phosphate-dependent degradation, showed that PNPase activity could be stalled in vivo and in vitro. Analysis of mutations in the SP82 moiety mapped the block to PNPase processivity to a particular stem-loop structure. This structure did not provide a block to processivity in the pnpA strain, suggesting that it was specific for PNPase. An abundant RNA with a 3' end located in the ermC coding sequence was detected in the pnpA strain but not in the wild type, indicating that this block is specific for a different 3'-to-5' exonuclease. The finding of impediments to 3'-to-5' degradation, with specificities for different exonucleases, suggests the existence of discrete intermediates in the mRNA decay pathway.
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PMID:Protection against 3'-to-5' RNA decay in Bacillus subtilis. 1057 37

Expression of the Bacillus subtilis glpD gene, which encodes glycerol-3-phosphate (G3P) dehydrogenase, is controlled by termination or antitermination of transcription. The untranslated leader sequence of glpD contains an inverted repeat that gives rise to a transcription terminator. In the presence of G3P, the antiterminator protein GlpP binds to glpD leader mRNA and promotes readthrough of the terminator. Certain mutations in the inverted repeat of the glpD leader result in GlpP-independent, temperature-sensitive (TS) expression of glpD. The TS phenotype is due to temperature-dependent degradation of the glpD mRNA. In the presence of GlpP, the glpD mRNA is stabilized. glpD leader-lacZ fusions were integrated into the chromosomes of B. subtilis and Escherichia coli. Determination of steady-state levels of fusion mRNA in B. subtilis showed that the stability of the fusion mRNA is determined by the glpD leader part. Comparison of steady-state levels and half-lives of glpD leader-lacZ fusion mRNA in B. subtilis and E. coli revealed significant differences. A glpD leader-lacZ fusion transcript that was unstable in B. subtilis was considerably more stable in E. coli. GlpP, which stabilizes the transcript in B. subtilis, did not affect its stability in E. coli. Primer extension analysis showed that the glpD leader-lacZ fusion transcript is processed differently in B. subtilis and in E. coli. The dominating cleavage site in E. coli was barely detectable in B. subtilis. This site was shown to be a target of E. coli RNase III.
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PMID:Different processing of an mRNA species in Bacillus subtilis and Escherichia coli. 1063 2

A recently described new signal for transcription termination in vitro by T7 RNA polymerase has now been tested in vivo. This signal, identified during transcription of the cloned human preproparathyroid hormone (PTH) gene, is also found in the phage T7 genome, at the concatemer junction (CJ). We introduced the 17-bp concatemer junction sequence at the ends of a test gene and control gene (both derived from T7 gene 9) in a T7 vector previously used to study effects of rare codons on expression. The CJ elements replaced the original vector's RNase III processing sites, and a new T7 promoter was also introduced to drive the downstream (control) gene. We assayed for test and control gene mRNA and protein by direct labeling with [32P]phosphate and [35S]methionine. The altered vector with CJ sequences (pCT1.1) expressed the upstream test gene, but showed poor expression of the downstream control gene. No discrete T7 mRNA bands could be discerned by direct labeling with 32P. A precursor vector with only the control gene in single copy expressed the protein much better, suggesting that the inhibition of control gene expression in pCT1.1 was a result of the upstream CJ element at the 3' end of the test gene. RT-PCR experiments were consistent with readthrough and possibly pausing at CJ. An RNA folding program predicts a highly stable secondary structure between the upstream CJ element and the control gene's translation start signals. These data support an interpretation that the CJ element is ineffective as a T7 transcription terminator in vivo in this vector, and that structure of the readthrough transcript blocks ribosome access to the downstream translation start. The readthrough transcripts are also likely to be less stable than properly terminated or processed T7 mRNA, because levels of test protein expression in pCT1.1 were reduced compared to original vector, and basal expression was negligible, while the original codon test vector shows substantial basal expression.
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PMID:The T7 concatemer junction sequence interferes with expression from a downstream T7 promoter in vivo. 1063 16


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