Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.9 (ribonuclease)
 6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The tRNATyr precursor molecule, synthesized from phi 80 psu3+ DNA (containing a single tRNA gene) by DNA-dependent RNA polymerase and q factor, was about 205 nucleotides long. The main product of its digestion with a ribonuclease tii preparation from Escherichia coli showed the same electrophoretic mobility as tRNAtyr precursor isolated in vivo and was found to be identical to it when analysed using fingerprint techniques. This intermediate precursor synthesized in vitro was converted further by processing with ribonuclease P into an RNA identical size to mature tRNATyr. It was concluded that the initiation of transcription of the tRNATyr gene in vitro occurs at the same site as that of transcription in vivo and a termination occurs at about 80 nucleotides beyond the CCA end of tRNATyr.
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PMID:Processing by ribonuclease II of the tRNATyr precursor of Escherichia coli synthesized in vitro. 32 7

The pyrE gene, encoding the pyrimidine biosynthetic enzyme orotate phosphoribosyltransferase, is the promoter distal gene of the dicistronic orfE-pyrE operon. The promoter proximal orfE gene, whose transcription and translation is important for regulation of the pyrE attenuator, encodes a 238-amino acid residue protein which was recently identified as the phosphorolytic ribonuclease, RNase PH, that removes nucleotides from the 3' ends of tRNA precursors. In this paper we report the construction of a plasmid, which overexpresses the orfE and pyrE gene products substantially, as well as the purification of the OrfE protein by ammonium sulfate precipitation and chromatography on phosphocellulose. The highly purified protein catalyzes the phosphorolytic cleavage of poly(A) at a rate of 1.6 mumol/min/mg and the formation of CDP from tRNA-CCA-Cn and orthophosphate at a rate equal to 0.14 mumol/min/mg, as characteristic for RNase PH. OrfE/RNase PH contains helix-turn-helix motifs resembling those in DNA-binding proteins, and it binds nonspecifically to DNA. On SDS gels, OrfE/RNase PH migrates as two distinct protein bands. This heterogeneity might be caused by post-translational modification other than proteolysis, or may be an electrophoretic artifact. The native protein is composed of two or more subunits.
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PMID:Overexpression and rapid purification of the orfE/rph gene product, RNase PH of Escherichia coli. 151 52

Rat liver mitochondria isolated in sucrose-N-tris(hydroxymethyl)methyl-2-aminoethane-sulphonic acid (TES) incorporated [(3)H]UTP into RNA for 1h. Incorporation was inhibited 50% by 1mug of actinomycin D/ml, 1mug of acriflavine/ml and 0.5mug of ethidium bromide/ml but was insensitive to rifampicin, rifamycin SV, streptovarcin and deoxyribonuclease. After the first 10min of incubation, the synthesis was insensitive to ribonuclease. RNA synthesis by mitochondria isolated in sucrose-EDTA was insensitive to actinomycin D and sensitive to ribonuclease during the first 10min of the incubation but thereafter the sensitivities were the same as for mitochondria isolated in sucrose-TES. In a hypo-osmotic medium the relative extent of incorporation of the four ribonucleoside triphosphates into RNA was CTP>UTP=ATP>>GTP. In an iso-osmotic medium the incorporation of CTP and GTP decreased. All four nucleotides were incorporated into RNA in a DNA-dependent process, as indicated by the inhibition by actinomycin D. In addition, CTP and ATP were incorporated into the CCA end of mitochondrial tRNA. ATP was also incorporated into an unidentified acid-insoluble compound, which hydrolysed in alkali to a product that was not ATP, ADP or 5'- or 2(3')-AMP. Atractyloside inhibited the incorporation of ATP into RNA with 50% inhibition at 2-3nmol/mg of protein. The [(3)H]UTP-labelled RNA had peaks of 16S and 13S characteristic of mitochondrial rRNA. In addition a peak at 20-21S was observed as well as heterogeneous RNA sedimenting throughout the gradient. The synthesis of all these species was inhibited by actinomycin D, indicating that rat liver mitochondrial DNA codes for mitochondrial rRNA as well as other as yet unidentified species.
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PMID:Synthesis of ribonucleic acid by isolated rat liver mitochondria. 440 94

The prr locus was originally described as coding a ribonuclease that is activated after phage T4 infection to cut within the anticodon of a specific tRNA, inactivating protein synthesis and thus blocking phage development. Wild-type T4 phage has two genes coding the enzymes polynucleotide kinase and RNA ligase, whose only function seems to be to repair the damage done by the anticodon nuclease. As the only apparent function of the prr ribonuclease is to combat phage infection, it can be considered as an RNA-based restriction enzyme. In non-infected cells, the prr enzyme is kept inactive in a complex with three other proteins which were predicted on the basis of DNA homologies to be the subunits of a type IC DNA restriction and modification system. Unlike other type IC systems so far characterized, prr is chromosomally rather than plasmid coded. However, sequences upstream from prr also have homology with sequences from the plasmid R124 and the prophage P1. We have now investigated the prr system and shown that it is indeed a bona fide type IC system which we call EcoprrI, and which is active both in vivo and in vitro. The system is fully functional even in the absence of the anticodon nuclease and seems to be a typical type I enzyme. EcoprrI recognizes the sequence CCA(N7)RTGC. One peculiarity is that, with low efficiency, EcoprrI will recognize and methylate variants of its recognition sequence such as CCT(N7)ATGC, which is methylated in one strand of the DNA only.
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PMID:The Escherichia coli prr region encodes a functional type IC DNA restriction system closely integrated with an anticodon nuclease gene. 814 41

In contrast to Escherichia coli, where all tRNAs have the CCA motif encoded by their genes, two classes of tRNA precursors exist in the Gram-positive bacterium Bacillus subtilis. Previous evidence had shown that ribonuclease Z (RNase Z) was responsible for the endonucleolytic maturation of the 3' end of those tRNAs lacking an encoded CCA motif, accounting for about one-third of its tRNAs. This suggested that a second pathway of tRNA maturation must exist for those precursors with an encoded CCA motif. In this paper, we examine the potential role of the four known exoribonucleases of B.subtilis, PNPase, RNase R, RNase PH and YhaM, in this alternative pathway. In the absence of RNase PH, precursors of CCA-containing tRNAs accumulate that are a few nucleotides longer than the mature tRNA species observed in wild-type strains or in the other single exonuclease mutants. Thus, RNase PH plays an important role in removing the last few nucleotides of the tRNA precursor in vivo. The presence of three or four exonuclease mutations in a single strain results in CCA-containing tRNA precursors of increasing size, suggesting that, as in E.coli, the exonucleolytic pathway consists of multiple redundant enzymes. Assays of purified RNase PH using in vitro-synthesized tRNA precursor substrates suggest that RNase PH is sensitive to the presence of a CCA motif. The division of labor between the endonucleolytic and exonucleolytic pathways observed in vivo can be explained by the inhibition of RNase Z by the CCA motif in CCA-containing tRNA precursors and by the inhibition of exonucleases by stable secondary structure in the 3' extensions of the majority of CCA-less tRNAs.
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PMID:Ribonuclease PH plays a major role in the exonucleolytic maturation of CCA-containing tRNA precursors in Bacillus subtilis. 1598 36

Unraveling the structure and assembly of the DNA packaging ATPases of the tailed double-stranded DNA bacteriophages is integral to understanding the mechanism of DNA translocation. Here, the bacteriophage phi29 packaging ATPase gene product 16 (gp16) was overexpressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the phi29 precursor capsid (prohead) to produce a packaging motor intermediate that was fully active in in vitro DNA packaging. The formation of higher oligomers of the gp16 from monomers was concentration dependent and was characterized by analytical ultracentrifugation, gel filtration, and electron microscopy. The binding of multiple copies of gp16 to the prohead was dependent on the presence of an oligomer of 174- or 120-base prohead RNA (pRNA) fixed to the head-tail connector at the unique portal vertex of the prohead. The use of mutant pRNAs demonstrated that gp16 bound specifically to the A-helix of pRNA, and ribonuclease footprinting of gp16 on pRNA showed that gp16 protected the CC residues of the CCA bulge (residues 18-20) of the A-helix. The binding of gp16 to the prohead/pRNA to constitute the complete and active packaging motor was confirmed by cryo-electron microscopy three-dimensional reconstruction of the prohead/pRNA/gp16 complex. The complex was capable of supercoiling DNA-gp3 as observed previously for gp16 alone; therefore, the binding of gp16 to the prohead, rather than first to DNA-gp3, represents an alternative packaging motor assembly pathway.
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PMID:DNA packaging motor assembly intermediate of bacteriophage phi29. 1867 82