Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protease-free bovine pancreatic deoxyribonuclease (DNase) (1.6 X 10(-4) mmol) was thiolated on the NH2 groups with N-acetyl-DL-homocysteine thiolactone (2.4 X 10(-2) mmol) at pH 10.5 with imidazole (2.4 X 10(-2) mmol) as the catalyst in the presence of 4,4'-dithiodipyridine (4.2 X 10(-2) mmol). The product obtained after 16 h at 4 degrees C, 2-acetamido-4-(4'-dithiopyridyl)butyryl-DNase, isolated by gel filtration, contained an average of 0.87 +/- 0.13 mol of mixed disulfide per mol of DNase. Ribonuclease (RNase) was thiolated in a similar manner, but under N2 in the absence of 4,4'-dithiodipyridine. The protein N-acetylhomocysteinyl-RNase contained on the average 0.94 +/- 0.11 mol of sulfhydryl groups per mol of RNase. The coupling of RNase ot DNase was accomplished by thiol-disulfide interchange at pH 6.2 and 25 degrees C for 90 min. The hybrid enzyme (yield 25--33%, based upon the DNase derivative used) was freed from unreacted DNase, RNase, and homodimers by gel filtration, affinity chromatography, and salting-out chromatography. The purified enzyme contained one molecule each of DNase and RNase and hydrolyzed thymus deoxyribonucleic acid (DNA) and yeast or transfer ribonucleic acid (RNA) with 75 and 40% of the efficiencies, respectively, of the parent enzymes. The RNA strand of the hybrid substrate, phage f1 DNA-[3H]RNA, prepared from phage DNA with RNA polymerase, was hydrolyzed rapidly by the hybrid enzyme but was not hydrolyzed by RNase alone. A conjugate of the two enzymes offers the possibility in vivo of delivering two enzymes that differ in size, charge, and biological function to the same site at the same time.
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PMID:Preparation of the bifunctional enzyme ribonuclease-deoxyribonuclease by cross-linkage. 48 31

A non-enzymic protein factor that increases the in vitro rate of synthesis by HeLa DNA polymerase alpha 15- to 30-fold with denatured DNA as template has been partially purified from the cytoplasmic fraction of HeLa cells. The stimulatory effect is highly specific for HeLa DNA polymerase alpha and for DNA templates that contain extensive regions of single-strandedness. Synthesis with denatured DNA as template presumably proceeds from 3'-hydroxyl termini formed at loop-back regions since the synthesized DNA product and template are covalently linked. The stimulatory protein factor chromatographs as a basic protein, has an approximate molecular weight of 30,000 daltons and binds with moderate affinity to denatured DNA cellulose, being eluted by o.4M NaCl. The purified factor lacks detectable DNA polymerase, exo- and endodeoxyribonuclease and RNA polymerase activities. It also does not promote helix-coil transitions with poly[d(A-T)] and Clostridium perfringens DNA.
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PMID:HeLa DNA polymerase alpha activity in vitro: specific stimulation by a non-enzymic protein factor. 64 10

Nuclei from seminal vesicle epithelium of adult guinea pigs were isolated in hypertonic sucrose solution. The incorporation of [3H]UTP by the isolated nuclei into acid-precipitable products was studied. Incorporation required ATP, GTP, CTP, UTP, and Mg+2. It was inhibited by addition of actinomycin D, deoxyribonuclease, or pyrophosphate to the reaction mixture. Thus, incorporation of [3H]UTP by isolated nuclei had the same characteristics that have been demonstrated for the reactions catalyzed by nuclear RNA polymerases. Using alpha-amanitin as a metabolic tool, we established concentrations of (NH4)2SO4. Mg+2, and nucleotides that give maximum assayable activities of nuclear RNA polymerases I and II. When the activities of polymerases I and II were measured in isolated seminal vesicle nuclei of guinea pigs that had been castrated 4 days earlier, a marked decrease in activities was found relative to control values (nuclei from intact animals). No further decrease was found 8 days after castration. Diminished accessibility to the nuclear DNA template and a decrease in the concentration of RNA polymerase molecules seemed to be responsible for the observed effects of castration on activities of RNA polymerases. An increase in ribonuclease activity did not seem to be responsible for the effects of castration. Activities of the enzymes did not change 2, 3, or 4 hours after intraperitoneal injection (2 mg/kg body weight) of each of five different androgens. Similarly, a single intraperitoneal injection of testosterone did not restore enzyme activity of polymerade I or II at any time during the first 24-hour period after hormone administration.
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PMID:RNA polymerase activities in isolated nuclei of guinea pig seminal vesicle epithelium: influence of castration and androgen administration. 90 9

The synthesis and template properties of T4 vegetative DNA were studied. The DNA-containing material in lysates of cells taken 20 min past T4 infection sediments in sucrose gradients as two major components. Both fractions function as templates for amino acid incorporation in a DNA-dependent in vitro system (coupled transcription-translation). The slower sedimenting activity is not present in uninfected cells and appears in wild type T4-infected cells only after 12 min at 30 degrees, shortly after DNA synthesis starts. It is dependent for its activity on an added S-30 fraction from either uninfected or T4-infected cells and is completely inhibited by deoxyribonuclease or rifampin. On a weight basis the slower sedimenting template is about 30 to 70% as active as mature T4 DNA when supplemented with S-30 extracts from uninfected cells. The spectrum of proteins synthesized in response to the slower sedimenting template is different from that produced in response to mature T4 DNA. In contrast to mature DNA, this template is capable of directing the synthesis of material that precipitates with antiserum directed against whole T4 particles. Thus, it appears capable of directing the synthesis of mRNA for phage structural proteins, i.e. late proteins. The faster sedimenting component is about 8-fold less active for stimulating amino acid incorporation than mature DNA. Significant amounts of RNA polymerase are associated with this DNA in active transcription complexes, yet polyacrylamide gel electrophoresis of the proteins synthesized in response to this fraction show a pattern that resembles the early proteins made from mature T4 DNA in extracts from uninfected cells.
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PMID:Template properties of bacteriophage T4 vegetative DNA. I. Isolation and characterization of two template fractions from gently lysed T4-infected bacteria. 110 12

1. Slow, spontaneous lysis of Halobacterium cutirubrum in 3 M-KCl yields DNA-dependent RNA polymerase as a complex with DNA that sediments completely at 45 000g. 2. Controlled deoxyribonuclease digestion of the complex, with or without subsequent sonication, releases the enzyme quantitatively in a soluble form that passes through ultrafilters with a molecular-weight exclusion limit of 50 000. 3. Purification of the active ultrafiltrate by gel filtration and hydroxyapatite chromatography gives a high yield of the purified alpha and beta subunits. 4. The low mol.wt. (17 800-19 000) of the soluble enzyme was confirmed by gel filtration and is unchanged by sonication of the DNA-enzyme complex. 5. A new assay applicable to both forms of the enzyme was developed. 6. The bivalent-cation requirement of the soluble form depends on the buffer concentration. 7. Both the DNA-enzyme complex and the low-molecular-weight soluble forms of the polymerase catalyse formation of short RNA chains only.
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PMID:The relationship between the deoxyribonucleic acid-bound and low-molecular-weight soluble forms of Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polymerase. 120 Sep 99

It is generally assumed that the machinery that transcribes genes is composed entirely of polypeptides. However, in vitro transcription by silkworm RNA polymerase III requires a transcription factor that is not a polypeptide. This component, TFIIIR, is distinct from the previously identified transcription components: RNA polymerase III, and the accessory factors TFIIIA, TFIIIB, TFIIIC, and TFIIID. The newly discovered TFIIIR is a macromolecule that appears to be composed of RNA. It is resistant to heat, detergent, phenol, protease, and deoxyribonuclease, but it is sensitive to alkali and ribonuclease.
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PMID:A class III transcription factor composed of RNA. 170 25

Transcription by purified mammalian RNA polymerase II in vitro leads to extensive formation of DNA-RNA hybrids between nascent RNA and the template DNA strand. This is especially clear during transcription of 3'-extended (dC-tailed) DNA templates where the nontranscribed DNA strand is progressively displaced as transcription proceeds [Kadesch, T. R., & Chamberlin, M. J. (1982) J. Biol. Chem. 257, 5286-5295]. Addition of small amounts of a HeLa cell extract to such a transcription system enhances renaturation of the template DNA and displacement of the nascent RNA, as measured by the sensitivity of the RNA to pancreatic ribonuclease. Using this latter assay, we have purified a protein factor (renaturase) 250-fold from HeLa cell extracts using chromatography on DEAE-cellulose, DNA-cellulose, and hydroxylapatite. Renaturase preparations facilitate complete renaturation of the template DNA duplex during transcription by RNA polymerase II and lead to concurrent displacement of the nascent RNA. Current preparations are free from all but traces of deoxyribonuclease or ribonuclease. The active component has a molecular weight of about 30000 as estimated by preparative density gradient sedimentation. We have examined the structure of transcribing RNA polymerase II complexes in the presence and absence of renaturase, using the electron microscope and the Williams polylysine technique [Williams, R. C. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2311-2315]. In the presence of renaturase, the DNA template is fully renatured, and a ternary complex in which the nascent RNA is displaced during transcription is seen.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Studies on transcription of 3'-extended DNA templates by mammalian RNA polymerase II. Partial purification and characterization of a factor from HeLa cells that facilitates renaturation of the DNA template. 399 13

Plagemann, Peter G. W. (Western Reserve University, Cleveland, Ohio), and H. Earle Swim. Replication of mengovirus. II. General properties of the viral-induced ribonucleic acid polymerase. J. Bacteriol. 91:2327-2332. 1966.-Mengovirus induces the appearance of a ribonucleic acid (RNA) polymerase activity in Novikoff hepatoma cells which is readily distinguished from the deoxyribonucleic acid (DNA)-dependent RNA polymerase since it is not inhibited by actinomycin D or deoxyribonuclease, but is inhibited by ammonium sulfate, and is stable at -17 C. The incorporation of uridine into RNA by infected cells in the presence of actinomycin D does not reflect the viral polymerase activity as measured in cell-free preparations. The viral-induced RNA polymerase is produced in a biphasic fashion. Puromycin inhibits the production of viral polymerase, and in its presence the enzyme appears to be unstable between 4 and 6 hr. Puromycin also prevents the secondary rise in polymerase which begins at the end of replicative cycle. Under these conditions, however, the polymerase appears to be stable. The overall data indicated that some unspecified process is responsible for the apparent instability of viral-induced RNA polymerase between 4 and 6 hr and that it becomes inoperative toward the end of the replicative cycle.
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PMID:Replication of mengovirus. II. General properties of the viral-induced ribonucleic acid polymerase. 428 86

Escherichia coli spheroplasts lysed by Brij 58 and deoxycholate were separated into supernatant (S) and membrane fractions by low-speed centrifugation. The membrane fraction was further divided into that which was releasable by deoxyribonuclease (fraction D) and that which was not (M). In the presence of 10(-2)m Mg(2+), the S, D, and M fractions contained, respectively, 60, 20, and 20% of the total cellular ribonucleic acid (RNA). Ribosomal and transfer RNA (rRNA, tRNA) were found in each fraction. The M + D fraction RNA was labeled more by a pulse label. Incorporation of uracil into the D fraction continued only as long as the uptake of exogenous uracil, suggesting that this was a major primary site of RNA synthesis. From pulse-labeled cells, each fraction contained precursor rRNA, and there was a 10S RNA in the M fraction. Ninety per cent of the ribosomal subunits and the ribosomal precursor particles, 26 and 43S, were in the S fraction. Precursor RNA (17S) was found in the 26S precursor particles. The D fraction contained 38% of the polysomes (this does not consider polysomes, if any, of the M fraction) which were labeled four times as much as the supernatant polysomes by a 1-min pulse of uracil. These results are interpreted to mean that new RNA is associated with a cytoplasmic membrane-RNA polymerase-DNA complex.
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PMID:"Compartmentalization" of Escherichia coli ribosomes and ribonucleic acid. 455 51

Lust, George (Fort Detrick, Frederick, Md.). Alterations of protein synthesis in arbovirus-infected L cells. J. Bacteriol. 91:1612-1617. 1966.-Cellular protein synthesis and ribonucleic acid (RNA) synthesis in mouse L cells were markedly depressed 1 hr after infection with Venezuelan equine encephalomyelitis virus. Host RNA and protein synthesis were inhibited more rapidly by the virus infection than by actinomycin D. In cells infected 4 hr, a cytoplasmic RNA polymerase was demonstrated which was absent in uninfected cells. At this time, deoxyribonucleic acid-directed RNA synthesis catalyzed by the nuclear RNA polymerase was inhibited in vitro in enzyme preparations from nuclei of virus-infected cells. For optimal activity, the cytoplasmic RNA polymerase required the four nucleoside triphosphates, Mg(++), and RNA. The enzyme was insensitive to actinomycin D and deoxyribonuclease, indicating that it catalyzed RNA-directed RNA synthesis. Attempts to purify the induced polymerase further were unsuccessful. Fresh preparations had to be used because the enzymatic activity was unstable.
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PMID:Alterations of protein synthesis in arbovirus-infected L cells. 592 79


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