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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19 + 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of 3H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115-120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19 + 2.1. Primers synthesized using M13mp19 + 2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.
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PMID:Pea chloroplast DNA primase: characterization and role in initiation of replication. 186 57

Pea chloroplast RNA polymerase has been obtained with about 2000-fold purification using DEAE-cellulose and phosphocellulose chromatography. The purified enzyme contained ten prominent polypeptides of 150, 130, 115, 110, 95, 85, 75, 48, 44 and 39 kDa and four other minor polypeptides of 90, 34, 32 and 27 kDa. Purification of this enzyme using chloroplast 16S rDNA promoter affinity column chromatography also yielded an enzyme with similar polypeptides. Purified polyclonal antibodies against the purified chloroplast RNA polymerase were found to recognize most of the polypeptides of the enzyme in Western blot experiments. Primary mobility shift of the 16S rRNA gene and ribulose-1,5-bisphosphate carboxylase large subunit (rbc-L) gene promoters observed with the chloroplast RNA polymerase was abolished by these antibodies. The specific in vitro transcription of these rRNA and mRNA genes was also inhibited by these antibodies. The transcription of the rRNA and mRNA genes was also abolished by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase. The chloroplast RNA polymerase was found to bind specifically to the chloroplast 16S rRNA gene promoter region as visualized in electron microscopy. The presence of the polypeptides of 130, 110, 75-95 and 48 kDa in the DNA-enzyme complex was confirmed by a novel approach using immunogold labeling with the respective antibodies. The polypeptides of this purified RNA polymerase were found to be localized in chloroplasts by an indirect immunofluorescence.
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PMID:Highly purified pea chloroplast RNA polymerase transcribes both rRNA and mRNA genes. 199 70

RNA polymerase 1 activity and nucleolar volume have been reported to increase in hepatocytes from rats fed a protein-free diet. Phosphorylation in vitro of a 110-kDa protein was enhanced in nuclei and nucleoli from livers of rats fed a protein-free diet. In nuclear extracts the 110-kDa protein in heat-treated nuclei was much more phosphorylated than from control liver. In contrast, casein kinase activity in the nuclear extract from control liver was comparable to that from livers of rats fed a protein-free diet. Nuclear extracts from control rat liver and livers of rats fed a protein-free diet were fractionated by DEAE-cellulose column chromatography. Casein kinase II (NII) eluted at around 0.17 M NaCl scarcely phosphorylates the 110-kDa protein. Chromatography of the nuclear extract from livers of rats fed a protein-free diet, but not from control liver, yielded fractions which eluted at 0.21-0.25 M NaCl and predominantly phosphorylated the 110-kDa protein. The phosphorylation of 110-kDa protein was not appreciably affected by a heparin concentration of 5 micrograms/ml, which completely inhibited casein kinase II. In addition, phosphorylation of the 110-kDa protein in liver nucleoli from rats fed a protein-free diet showed a lower sensitivity to heparin than that in control rat liver nucleoli. These results suggest that enhanced phosphorylation of the nuclear 110-kDa protein in livers from rats fed a protein-free diet is due to the induction of a 110-kDa protein kinase distinct from casein kinase II.
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PMID:Enhanced phosphorylation of a nucleolar 110-kDa protein in rat liver by dietary manipulation. 216 18

The polypeptide encoded by the vaccinia virus open reading frame D7R was synthesized in bacteria. Immunization of rabbits with the polypeptide resulted in antibodies that specifically recognized a virion polypeptide of 20,000 daltons. The immunoreactivity with the 20,000-dalton polypeptide was found to coincide with the virion-associated DNA-dependent RNA polymerase through DEAE-cellulose chromatography and glycerol gradient sedimentation. These results argue that the product of the vaccinia open reading frame D7R is a subunit of the viral RNA polymerase.
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PMID:Vaccinia virus gene D7R encodes a 20,000-dalton subunit of the viral DNA-dependent RNA polymerase. 221 12

Nuclei were isolated from bloodstream forms of Trypanosoma brucei by nitrogen cavitation and sedimentation through percoll density gradients. Transcription studies with these nuclei in vitro demonstrated features not seen with other eukaryotes: RNA synthesis was much greater in the presence of Mn2+ than with Mg2+ and was sensitive to high concentrations (10-100 micrograms/ml) of alpha-amanitin at all salt concentrations tested (25-300 mM ammonium sulphate). RNA polymerase extracted from nuclei by sonication at high ionic strength chromatographed as a single peak, sensitive to high alpha-amanitin concentrations, on DEAE-sephadex under conditions which resolved the classic three RNA polymerase forms when rat liver nuclear extracts were used.
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PMID:Unusual RNA polymerase content of Trypanosoma brucei nuclei. 241 50

Previous studies suggested multiple sites of action of aflatoxin B1 (AFB1) in vivo to inhibit rat liver nuclear RNA synthesis--it impairs nucleolar DNA template function and inhibits RNA polymerase II activity. We have previously shown that AFB1 activated in vitro inhibits nucleolar RNA synthesis. The question is whether AFB1 can inhibit RNA polymerase II under these in vitro conditions. Male Sprague-Dawley rats, 200 g, were injected i.p. with 0.6 mg AFB1 and liver nuclei were isolated 2 h later. When the total nuclear free RNA polymerases were extracted and assayed in the absence and presence of alpha-amanitin (3.2 micrograms/ml), we found that only alpha-amanitin-sensitive (i.e., RNA polymerase II) activity was inhibited (97%). DEAE-Sephadex chromatography confirmed this result. When total nuclear free RNA polymerases were incubated with AFB1 activated in vitro under conditions producing 70% inhibition of nucleolar RNA synthesis, no inhibition was observed for either alpha-amanitin-sensitive or -resistant activities. Similar results were obtained with low and high (28 and 167 micrograms/ml) concentrations of AFB1. This was further confirmed using highly purified RNA polymerase II. We conclude that AFB1 inhibition of RNA polymerase II activity in vivo is not a result of direct interaction of AFB1 to the enzyme.
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PMID:Evidence for an indirect mechanism of aflatoxin B1 inhibition of rat liver nuclear RNA polymerase II activity in vivo. 241 4

Cytoplasmic extracts made from HeLa cells that have been harvested late after infection with vaccinia virus are capable of specifically transcribing templates containing vaccinia virus late-gene promoters. We applied such an extract to a phosphocellulose column and eluted the proteins with a series of buffers containing successively higher concentrations of NaCl. None of three column fractions alone was capable of specific transcription of a late-gene template. However, specific transcriptase activity could be reconstituted by mixing column fractions, with maximal activity seen when all three fractions were present. The activities present in all fractions were heat labile, resistant to micrococcal nuclease, and present only in extracts from vaccinia virus-infected cells. A quantitative complementation assay was used to further purify one factor, named VLTF-1, over subsequent columns of DEAE-cellulose and hydroxylapatite. VLTF-1 was separated from endogenous RNA polymerase, was a late-promoter-specific transcription factor, and had a sedimentation rate consistent with an apparent Mr of 45,000. The RNA polymerase-containing fraction was not only necessary for transcription with a late-promoter template but alone was capable of specifically transcribing a vaccinia virus early-gene promoter. A further difference between early and late gene transcription in this system was in the ability of the ATP analog beta-8-imidoadenosine-5'-triphosphate (AMP-PNP) to substitute for ATP in supporting specific transcription of only the late-promoter template. The system reconstituted from the various fractions retained the ability to produce the novel poly(A) sequence found on the 5' end of vaccinia virus late messages.
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PMID:Identification of factors specific for transcription of the late class of vaccinia virus genes. 247 68

Benzo[a]pyrene was administered intraperitoneally to male Wistar rats as a single dose of 20 mg.kg-1 body weight. Two hours after its administration, livers were excised and the enzyme RNA polymerase was solubilized from purified nuclei. The enzyme was resolved into three forms, polymerase I, II and III by partial purification on DEAE-Sephadex A-25 column. All the three forms of nuclear RNA polymerase were inhibited in response to administration of benzo[a]pyrene. This was evident in terms of total yield as well as specific activity of each enzyme. The inhibition of nuclear transcription following administration of benzo[a]pyrene as observed previously (1) along with the present results on the enzyme inhibition clearly suggest that benzo[a]pyrene acts on both the major components of transcription machinery i.e. the template chromatin and the enzyme RNA polymerases.
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PMID:Alteration in hepatic nuclear RNA polymerase activity following benzo[a]pyrene administration in rat. 251 39

Erwinia carotovora RNA polymerase consists of the holoenzyme structure sigma 2 beta beta' sigma as found in Escherichia coli and other bacteria. E. carotovora RNA polymerase can synthesize RNA using lambda, T7 of T4 DNA as templates; however, it is two times less active on these templates and is more temperature-sensitive than the E. coli enzyme. The alpha subunit of the E.. carotovora enzyme is lower in molecular mass than its E. coli counterpart. The sigma factors from E. coli and E. carotovora are similar in size and in their ability to stimulate RNA synthesis by core enzyme on DNA templates such as T7 DNA. An additional protein of 115 000 Da molecular mass, termed gamma, is found associated with E. carotovora RNA polymerase. The gamma protein is tightly associated with the polymerase subunits as it is not dissociated by gel filtration in buffer containing 0.5 M NaCl. It can be purified by passing the Agarose 1.5 m enzyme through coupled Bio-Rex 70 and DEAE-cellulose columns. The gamma-protein, when present in excess over the sigma subunit, inhibits holoenzyme activity on T7 DNA but not on poly[d(A-T)]and may thus interfere with sigma activity. The gamma protein by itself cannot transcribe T7 DNA or poly[d(A-T)], nor does it stimulate core enzyme activity on T7 DNA. E. carotovora rho has a subunit molecular mass of 48 000 Da and exhibits RNA-dependent phosphohydrolysis of adenosine ribonucleoside triphosphate. E. coli and E. carotovora rho are indistinguishable immunologically, as total fusion of precipitin bands is observed. E. carotovora rho elutes from a phosphocellulose column at a salt concentration of about 0.21 M KCl, compared to that of 0.29 M KCl for E. coli rho. The poly(C)-dependent ATPase activity of E. carotovora rho is more-temperature sensitive and is six to ten times less active than that of E. coli rho. E. carotovora rho is capable of terminating RNA transcripts, as indicated by a decrease in RNA synthesis using lambda or T7 DNA as template and E. carotovora or E. coli polymerase as the transcribing-enzyme.
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PMID:Purification of RNA polymerase and transcription-termination factor Rho from Erwinia carotovora. 257 93

Mammalian cells contain two forms of RNA polymerase II, designated IIO and IIA, that differ in the extent of phosphorylation within the C-terminal domain of their largest subunit. Phosphorylation of this domain, which results in the conversion of RNA polymerase IIA to IIO, may play an important role in the transition from the initiation to the elongation phase of transcription. A third form of the enzyme, RNA polymerase IIB, is found in vitro and lacks the repetitive C-terminal domain. Purified calf thymus RNA polymerase IIA was labeled selectively with casein kinase II in the presence of [gamma-32P]ATP and used as substrate for the identification and partial purification of factors that catalyze the conversion of RNA polymerase IIA to IIO. HeLa cell S-100 transcription extracts contain such an activity that cofractionates with factors essential for promoter-dependent transcription through heparin-Sepharose, DEAE-5PW, and DE52 chromatography. The activity is dependent on either ATP, GTP, or dATP, requires a hydrolyzable beta,gamma-phosphoanhydride bond, and cannot utilize pyrimidine nucleoside triphosphates. This observation supports the idea that the conversion activity is a protein kinase. Transcription of the major late promoter of adenovirus-2 was carried out in the presence of a reconstituted transcription extract containing purified RNA polymerases IIO, IIA, or IIB, and the nature of the elongating enzyme was determined by photoaffinity labeling. When the reaction was initiated with RNA polymerase IIO or IIB, nascent transcripts were found cross-linked to subunit IIo or IIb, respectively. However, when the reaction was initiated with RNA polymerase IIA, nascent transcripts were cross-linked to subunit IIo. Consequently, phosphorylation of the C-terminal domain of subunit IIa must have occurred prior to elongation. The copurification of RNA polymerase IIA to IIO conversion activity with factors essential for promoter-dependent transcription and the observation that RNA polymerase II containing an unphosphorylated C-terminal domain is phosphorylated prior to elongation suggest that protein kinases that phosphorylate the C-terminal domain of subunit IIa may play an essential role in transcription.
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PMID:The transition of RNA polymerase II from initiation to elongation is associated with phosphorylation of the carboxyl-terminal domain of subunit IIa. 258 85


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