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)

We report experiments designed to elucidate the mechanism by which RNA polymerase advances from the open complex to synthesis of a stably bound RNA chain during transcription initiation. Techniques used include deoxyribonuclease I footprinting, methylation protection, and exonuclease III digestion through upstream domains, each applied to the open, abortive and productive transcription complexes of Escherichia coli RNA polymerase with the lac promoter. The results show a slight loss of upstream open complex contacts during abortive transcription of a 6-mer and 8-mer, but a large loss of these contacts upon escape from abortive cycling into productive transcription at the 11-mer. We propose a model for early initiation in which competition between open complex polymerase-DNA contacts on one hand and initiated complex polymerase-DNA-RNA interactions on the other produces a "stressed intermediate" during formation of a short RNA-DNA duplex. The strain energy is relieved either by ejecting the short RNA, resulting in aborted initiation, or by eliminating the sigma subunit and breaking the open complex contacts, thereby escaping abortive cycling into productive transcription. Further evidence for this model is based on the observation that destabilization of interactions specific for either open complex or initiated complex has the predicted effect on the amount of abortive cycling. The model predicts a complicated relationship between overall promoter strength and DNA sequence changes that alter polymerase-DNA interactions.
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PMID:A stressed intermediate in the formation of stably initiated RNA chains at the Escherichia coli lac UV5 promoter. 243 94

The human ribosomal RNA promoter contains two distinct control elements (UCE and core) both of which are recognized by the sequence-specific DNA binding protein UBF1, which has now been purified to apparent homogeneity. The purified factor activates RNA polymerase I (RNA pol I) transcription through direct interactions with either control element. A second RNA pol I transcription factor, designated SL1, participates in the promoter recognition process and is required to reconstitute transcription in vitro. Although SL1 alone has no sequence-specific DNA binding activity, deoxyribonuclease I footprinting experiments reveal that a cooperative interaction between UBF1 and SL1 leads to the formation of a new protein-DNA complex at the UCE and core elements. In vitro transcription experiments indicate that formation of the UBF1-SL1 complex is vital for transcriptional activation by UBF1. Thus, protein-protein interactions between UBF1 and SL1 are required for targeting of SL1 to cis-control sequences of the promoter.
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PMID:Functional cooperativity between transcription factors UBF1 and SL1 mediates human ribosomal RNA synthesis. 341 83

Deoxynucleotides have been incorporated into RNA synthesized in vitro by RNA polymerase with either double-stranded or single-stranded DNA as a template. By use of this technique to block or promote cleavage at a particular phosphodiester bond, a variety of specific cleavages may be obtained with the available ribonucleases and deoxyribonuclease I. These methods should greatly increase the ease and rapidity of nucleotide sequence determinations.
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PMID:Deoxysubstitution in RNA by RNA polymerase in vitro: a new approach to nucleotide sequence determinations. 461 37

Extremely mild treatment with micrococcal nuclease of isolated nuclei yields subnuclear fractions in which the majority of RNA polymerase II transcriptional complexes formed in vivo are segregated [Tata & Baker (1978) J. Mol. Biol. 118, 249-272]. We now describe different approaches followed to established whether or not the nuclei are thus resolved into transcribed and non-transcribed DNA. First, we have compared the sensitivity to deoxyribonuclease I, which is known to digest preferably expressed genes as present in nuclei or chromatin, of three micrococcal-nuclease-derived fractions from nuclei of different transcriptional activities. In transcriptionally active nuclei (rat liver, hen liver and oviduct, and Xenopus liver), the DNA in a polynucleosomal fraction comprising 6-15% of DNA and the majority of template-engaged RNA polymerase II (fraction P2) was 10-50 times as sensitive to deoxyribonuclease I as the DNA in the other two fractions (fractions P1 and S, comprising 78-88% of total nuclear DNA as large polynucleosomal aggregates and 2-6% of DNA mostly as mononucleosomes, respectively). In transcriptionally inactive nuclei obtained from hen erythrocytes, micrococcal nuclease did not separate DNA into fractions exhibiting such differential sensitivities. Second, we have monitored the partition of an expressed gene. Hybridization of complementary DNA to Xenopus albumin mRNA revealed a 5-10-fold enrichment of the albumin (but not the globin) gene in the P2 fraction of nuclei from Xenopus liver in which this gene is fully expressed. Third, a large part of the nascent rapidly labelled RNA synthesized in vivo in rat liver nuclei was recovered in the micrococcal-nuclease-derived fraction that is more susceptible to digestion with deoxyribonuclease I. It is concluded that mild micrococcal-nuclease treatment of nuclei causes their separation into transcribed and non-transcribed DNA as determined by a number of very different criteria.
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PMID:Subnuclear fractionation by mild micrococcal-nuclease treatment of nuclei of different transcriptional activities causes a partition of expressed and non-expressed genes. 615 73

Cardiac hypertrophy in spontaneously hypertensive rats is associated with increased nuclear RNA polymerase activity. In order to explore mechanisms facilitating the interaction of the enzyme with its endogenous template, we compared the structure of nuclear chromatin from myocytes of 20-week-old spontaneously hypertensive rats and normotensive Wistar-Kyoto controls. Enhanced RNA synthesis in hypertensive rats was accompanied by increased susceptibility to digestion by deoxyribonuclease I. Nick translation of nuclei also resulted in higher nucleotide incorporation in hypertensive rats. Salt-extraction abolished the differences in deoxyribonuclease I sensitivity between the two animal groups. Reconstitution with either 0.35 M NaCl-extract or high mobility group (HMG) non-histone proteins restored digestion susceptibility but did not equalize SHR and WKY cells. SDS-polyacrylamide gel electrophoresis of 0.35 M NaCl-extracts and supernatants from deoxyribonuclease I digestion revealed the presence of HMG proteins which were preferentially released in hypertensive rats. There was a small but statistically significant increase in nuclear HMG protein content in hypertensive rats (0.12 +/- 0.02 mg/mg DNA vs. 0.09 +/- 0.02 mg/mg DNA in Wistar-Kyotos, P less than 0.05) but no difference in their electrophoretic appearance. These results indicate that chromatin structure is altered in the hypertrophied myocardium with resultant increase in deoxyribonuclease I susceptibility. This increase appears to be partly dependent on the high-mobility group non-histone proteins.
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PMID:Enhanced myocardial RNA synthesis in spontaneously hypertensive rats possible role of high-mobility group non-histone proteins. 617 42

The presence of actin in eukaryotic nuclei, and, especially, its functional significance has not been well established. We have found that under routine immunocytochemical conditions, no actin can be detected in insect follicle cell nuclei by means of antibody (both mono- and polyclonal) or phalloidin staining. However, a pretreatment of nuclear preparations with two different endonucleases (deoxyribonuclease I or micrococcal nuclease) to remove a substantial amount of chromosomal DNA uncovers the presence of nuclear actin for both antibody and phalloidin detection. Employing the same nuclease digestion followed by antibody or phalloidin staining with squash preparations of Drosophila polytene chromosomes revealed that the nuclear actin is directly associated with the chromosomes. A strong positive signal in the polytene chromosomes obtained with phalloidin labeling not only confirmed the presence of actin in the chromosomes, but indicates that a considerable amount of nuclear actin is present in filamentous form (F-actin) rather than monomeric (G-actin). The detection of actin associated with Xenopus embryo chromosomes suggests the significance of chromosomal actin for diploid vertebrate cells. Using the specific actin disrupting agent cytochalasin D, we have demonstrated the structural significance of nuclear actin in maintaining the linear integrity of polytene chromosomes. Further, we present evidence that RNA polymerase II closely interacts with the chromosomal actin scaffold, and that its association with chromosomes does not require the presence of DNA.
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PMID:An actin infrastructure is associated with eukaryotic chromosomes: structural and functional significance. 752 80

The Ku autoantigen is a heterodimer of 70 kDa (p70) and -80 kDa (p80) subunits that is the DNA-binding component of a DNA-dependent protein kinase (DNA-PK). The 350 kDa (p350) catalytic subunit of DNA-PK phosphorylates Sp-1, Oct-1, p53 and RNA polymerase II in vitro, but the precise cellular role of DNA-PK remains unclear. In the present studies, the assembly of p70/p80 heterodimers and the interaction of Ku with DNA was investigated using recombinant vaccinia viruses directing the synthesis of human p70 (p70-vacc) and p80 (p80-vacc), and monoclonal antibodies (mAbs). Expression of human Ku antigens in rabbit kidney (RK13) cells could be demonstrated by immunofluorescent staining because this cell line contains little endogenous Ku. A novel mAb designated 162 stained the nuclei of RK13 cells coinfected with p70-vacc and p80-vacc, but not cells that were infected with either virus alone, suggesting that it recognized the p70/p80 heterodimer but not monomeric p70 or p80. In agreement with the immunofluorescence data, 162 immunoprecipitated both p70 and p80 from extracts of coinfected cells, but did not immunoprecipitate either subunit by itself from extracts of cells infected with p70-vacc or p80-vacc, respectively. Conversely, the binding of 162 to Ku isolated from human K562 cells stabilized the p70/p80 heterodimer under conditions that normally dissociate p70 from p80. The nuclei of cells infected with p70-vacc alone could be stained with mAb N3H10 (anti-p70) and cells infected with p80-vacc alone could be stained with mAb 111 (anti-p80), indicating that the formation of p70/p80 heterodimers was not required for nuclear transport. Finally, free recombinant and cellular p70 both bound to DNA efficiently in vitro, suggesting that free p70, like the p70/p80 heterodimer, serves as a DNA-binding factor. Moreover, free human p70 could be released from the nuclei of p70-vacc-infected RK13 cells by deoxyribonuclease I treatment, suggesting that it was associated with chromatin in vivo. The nuclear transport of free p70 and the association of free p70 with chromatin in vivo raise the possibility that newly synthesized cellular p70 might undergo nuclear transport and DNA-binding prior to dimerization with p80 or assembly with p350.
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PMID:Assembly and DNA binding of recombinant Ku (p70/p80) autoantigen defined by a novel monoclonal antibody specific for p70/p80 heterodimers. 769 19

Elongation complexes of RNA polymerase II, RNA-DNA-enzyme ternary complexes, are intermediates in the synthesis of all eukaryotic mRNAs and are potential regulatory targets for factors controlling RNA chain elongation and termination. Analysis of such complexes can provide information concerning the structure of the catalytic core of the RNA polymerase and its interactions with the DNA template and RNA transcript. Knowledge of the structure of such complexes is essential in understanding the catalytic and regulatory properties of RNA polymerase. We have prepared and isolated complexes of purified RNA polymerase II halted at defined positions along a DNA template, and we have used deoxyribonuclease I (DNAse I) to map the interactions of the polymerase with the DNA template. DNAse I footprints of three specific ternary complexes reveal that the enzyme-template interactions of individual elongation complexes are not identical. The size of the protected region is distinct for each complex and varies from 48 to 55 bp between different complexes. Additionally, the positioning of the protected region relative to the active site varies in different complexes. Our results suggest that RNA polymerase II is a dynamic molecule and undergoes continual conformational transitions during elongation. These transitions are likely to be important in the processes of transcript elongation and termination and their regulation.
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PMID:Contacts between mammalian RNA polymerase II and the template DNA in a ternary elongation complex. 844 6

Glucocorticoids inhibit transcription of the murine cytoplasmic thymidine kinase gene (Tk-1). Glucocorticoid regulation of Tk-1 transcription can be demonstrated in cells that are arrested in late G1. This observation indicates that inhibition of Tk-1 expression is not dependent upon redistribution within the cell cycle but is due to glucocorticoid regulation of this gene. Transfection studies have been carried out using chimeric genes in which restriction fragments of the Tk-1 promoter were fused to chloramphenicol acetyltransferase or neomycin phosphotransferase. These chimeric reporters were assayed for stable expression and glucocorticoid regulation in P1798 lymphoma cells. A 140-bp fragment, extending from -143 to -3 bp with respect to the thymidine kinase translational start site, was capable of both basal and glucocorticoid-regulated transcription of reporter genes. The extent of inhibition by glucocorticoids was similar to that observed for the endogenous gene, and no increase in basal expression or the extent of inhibition was observed with constructs containing additional 5'-flanking DNA. The 140-bp Tk-1 core promoter fragment binds to transcription factors in extracts from P1798 cells. Control cell extracts contain factors that bind to and protect (from deoxyribonuclease I) a distal promoter element from -106 to -87 bp, relative to the translational start site. A second, proximal element was protected at -43 to -36 bp. The proximal element of the Tk-1 promoter resembles an RNA polymerase II initiator element. No other elements were protected. Glucocorticoids inhibit the amount or activity of the transcription factor that binds to this initiator-like element within the Tk-1 promoter. This element, when fused to upstream activation sequences from the herpes simplex virus thymidine kinase promoter, conveys glucocorticoid sensitivity in cis.
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PMID:Glucocorticoid regulation of a transcription factor that binds an initiator-like element in the murine thymidine kinase (Tk-1) promoter. 896 Dec 64

We found and isolated two natural products in the extract from a basidiomycete, Ganoderma lucidum, as eukaryotic DNA polymerase inhibitors. The compounds were identified as cerebrosides, (4E,8E)-N-D-2'-hydroxypalmitoyl- 1-O-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine and (4E,8E)-N-D-2'-hydroxystearoyl-1-O-beta-D-glucopyranos yl-9-methyl- 4,8-sphingadienine and were found to be identical to the mushroom fruiting body-inducing substances (FIS) reported. These cerebrosides selectively inhibited the activities of replicative DNA polymerases, especially the alpha-type, from phylogenetically broad eukaryotic species, whereas they hardly influenced the activities of DNA polymerase beta, prokaryotic DNA polymerases, terminal deoxynucleotidyl transferase, HIV reverse transcriptase, RNA polymerase, deoxyribonuclease I, and ATPase. The inhibition of another replicative polymerase, the delta-type, was moderate. The inhibitions of the replicative polymerases were dose-dependent, and the IC50 for animal or mushroom DNA polymerase alpha was achieved at approximately 12 micrograms/ml (16.2 microM) and for animal DNA polymerase delta at 57 micrograms/ml (77.2 microM). FIS is possibly a DNA polymerase inhibitor specific to the replicative enzyme group, and the fruiting body formation may be required for the suppression of the DNA replication or the vegetative growth of the mycelium.
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PMID:A mushroom fruiting body-inducing substance inhibits activities of replicative DNA polymerases. 970 23


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