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)

The accessibility of extracellular and nuclear simian virus 40 (SV40-M and SV40-I, respectively) virion chromatin DNAs to micrococcal nuclease, DNase I, BglI, EcoRI, and RNA polymerase was examined. Our results support the following conclusions: (i) the intranucleosomal DNA of SV40-I chromatin, similar to the precursor 75S chromatin complex, is resistant to enzymatic activity; and (ii) SV40-M virion chromatin is modified in a manner which increases the accessibility of viral DNA to enzymes, and the distinction between nucleosomal DNA and linker DNA is absent. Micrococcal nuclease digestion of SV40-I virion chromatin gave a typical nucleosomal DNA ladder pattern with a repeat unit of 205 base pairs of DNA. SV40-I chromatin was sensitive to cleavage with endonuclease BglI, but not with EcoRI. When SV40-I virion chromatin was used as a template, the rate of incorporation of ribonucleoside triphosphates into RNA was 5% of that obtained with naked form SV40 form I DNA. Micrococcal nuclease digestion of SV40-M virion chromatin resulted in submonomeric DNA fragments of approximately 55 base pairs, but no larger repeating unit of DNA was observed. SV40-M virion chromatin was sensitive to cleavage with either BglI or EcoRI and was approximately 20% more susceptible to digestion with DNase I than was SV40-I virion chromatin. The transcriptional efficiency of the extracellular virion chromatin was almost equivalent to that of naked SV40 form I DNA and was 16-fold higher than the rate observed with nuclear virion chromatin. The increased transcriptional activity was dependent upon the presence of nonhistone viral protein VP1 or VP2 or both.
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PMID:Simian virus 40 maturation: chromatin modifications increase the accessibility of viral DNA to nuclease and RNA polymerase. 626 46

Biochemical mapping experiments of the simian rotavirus SA11 genome were performed to determine which double-stranded RNA segment coded for each of the viral polypeptides. Viral RNA transcripts were synthesized in vitro by using the endogenous viral RNA polymerase and fractionated by electrophoresis in acid-urea agarose gels. The fractionated transcripts were translated in two cell-free systems: micrococcal nuclease-treated reticulocyte lysates and wheat germ extracts. The polypeptide products were identified by polyacrylamide gel electrophoresis and partial peptide analysis and compared with polypeptides synthesized in infected cells or found in purified virus. The RNA segment that coded for each transcript was determined by hybridization of the fractionated transcripts to the double-stranded RNA genome and analysis of the hybrids by electrophoresis in polyacrylamide gels. Primary gene products were assigned for 10 of the rotavirus transcripts and 10 of the double-stranded RNA segments. The coding assignments are as follows: the inner-capsid polypeptides, VP1, VP2, and VP6, were assigned to segments 1, 2, and 6, respectively; the major outer-capsid polypeptides, VP3 and VP7, were assigned to segments 4 and 9, respectively; segments 5, 7, and 8 coded for nonstructural polypeptides with molecular weights of 53,000, 34,000, and 35,000, respectively; segment 10 coded for the 20,000-molecular-weight precursor to the 29,000-molecular-weight glycosylated nonstructural polypeptide; and segment 11 coded for a 26,000-molecular-weight polypeptide that may be the precursor to the minor outer-capsid polypeptide VP9. Several methods were used to determine the product of gene segment 3, and the problems associated with the identification of this gene product are discussed.
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PMID:Biochemical mapping of the simian rotavirus SA11 genome. 630 11

The ribosomal genes of Dictyostelium discoideum are extrachromosomal palindromic DNA molecules situated in the nucleolus. Each molecule comprises ribosomal RNA coding regions and non-transcribed spacer regions. We used both biochemical and electron microscopic approaches to investigate the structure of transcribing and non-transcribing chromatin. Nucleoli from exponentially growing cells were digested with micrococcal nuclease, and the resulting DNA fragments were separated by gel electrophoresis and transferred to DBM paper. They were hybridized with cloned EcoRI fragments derived from different parts of the ribosomal gene. Probes of the coding region showed a smear, while probes of the non-transcribed regions gave pronounced banding patterns more complex than typical nucleosome repeats, but not due solely to sequence-specific cutting by micrococcal nuclease. The DNA of the coding region was digested more quickly than that of the non-transcribed ones. When nucleoli were digested with restriction enzymes, sites within the coding region were accessible and sites in the non-transcribed region were protected. The structure of ribosomal chromatin in differentiating cells, in which the rate of ribosomal RNA synthesis is reduced, was examined using essentially the same methods. The coding region, probed by hybridization to micrococcal digests, then showed a typical DNA repeat pattern indicating that this region had become condensed into nucleosomes, and its accessibility to restriction enzymes was very much reduced. On electron micrographs of lysed nucleoli from exponentially growing cells, two types of chromatin were observed, one with a beaded nucleosomal appearance, the other with putative RNA polymerase molecules attached to fibres indistinguishable from free DNA adsorbed to the same grid. The combined results suggest that whereas regions that are not transcribed are packaged with proteins that protect them from nuclease digestion, actively transcribing ribosomal genes are associated with few macromolecular constituents apart from those required for transcription and its regulation.
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PMID:Chromatin structure along the ribosomal DNA of Dictyostelium. Regional differences and changes accompanying cell differentiation. 630 25

A large body of circumstantial evidence indicates that receptors located in nuclei of T3 responsive tissues represent a site of initiation of thyroid hormone action at the cellular level. Partial characterization of T3 receptors indicates that these proteins are monomeric structures in nuclei and are chromatin-associated non-histone proteins. Treatment of rat liver nuclei with either pancreatic DNase I or micrococcal nuclease releases T3 receptors from nuclei in two forms: a predominant (95 400 Mr; 5.5-6.0S) and a minor (265 000-365 000 Mr; 12.5S) nucleoprotein complex. Similar structures are excised from rat kidney, brain, and heart nuclei and from GH1 pituitary cell nuclei by micrococcal nuclease digestion. These endonuclease-excised receptor-containing complexes are significantly larger than the salt-extracted receptor (50 000 Mr; 3.5S). The presence of DNA and other non-receptor proteins in these structures indicates that T3 receptors probably function within multimeric complexes in vivo. Although T3 receptors appear to be associated with DNA between nucleosomes, i.e. linker DNA, it is not entirely clear whether all or only a fraction of T3 receptors interact with nucleosomal components. The 12.5S receptor-containing nucleoprotein complex may represent T3 receptors in association with linker DNA and nucleosomal components. T3 receptors do not appear to be uniformly distributed to all chromatin fractions, but are associated with structures having characteristics of transcriptionally active chromatin. They are found in a region of chromatin which is enriched in RNA polymerase activity, rapidly labeled RNA and non-histone proteins, and depleted of histone Hl. This region is also highly sensitive to both micrococcal nuclease and pancreatic DNase I digestion. The association of receptors with transcriptionally active chromatin, however, must be considered provisional until additional details of the precise receptor-chromatin interaction have been established. The recent demonstration of a 20-fold increase in a specific hepatic mRNA four hours following administration of T3 to hypothyroid rats indicates that thyroid hormone potentially has very rapid effects on hepatic gene expression. However, significant changes in nuclear protein phosphorylation, nuclear protein composition, and chromatin structure have not been detected within this four-hour period. Thus, effects of T3 on hepatic gene expression are brought about by local and presumably subtle changes in nuclear function.
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PMID:Association of thyroid hormone receptors with chromatin. 631 18

A protein factor which stimulates DNA polymerase alpha activity on heat-denatured DNA has been purified from mouse FM3A cells. The final preparation had a specific activity of 43,000 units/mg protein and lacked detectable DNA polymerase, RNA polymerase, DNA-dependent- and independent ATPase, exo- and endodeoxyribonuclease and phosphatase activities. The stimulating factor sedimented at 2.9S in a glycerol gradient. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the glycerol gradient fraction revealed the presence of a major band of 36,000 daltons, the amount of which corresponded well with the level of stimulating activity. The stimulation by the factor was specific for heat-denatured DNA, and a little or no stimulation was observed with native DNA, ribo- and deoxyribohomopolymers and single stranded circular DNA. Alkaline sucrose gradient sedimentation analysis of the reaction products revealed that newly synthesized DNA was covalently linked to the termini of heat-denatured DNA. The average chain length of the elongated span determined by the digestion with micrococcal nuclease and phosphodiesterase II, did not differ between in the presence and absence of the stimulating factor, suggesting that the stimulation by the factor was due to the increase in the initiation frequency of DNA synthesis from the 3'-hydroxyl terminus of heat-denatured DNA.
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PMID:Purification and characterization of a factor stimulating DNA polymerase alpha activity from mouse FM3A cells. 632 2

When isolated HeLa cell nuclei were preincubated under transcription conditions with excess E. coli RNA polymerase, chromatin DNA became relatively resistant to digestion by micrococcal nuclease. Quantitation of the DNA content in nuclei after enzyme digestion revealed that approximately twice as much nuclease was required to give the same levels of release of DNA fragments from transcribed as from untranscribed nuclei. Resistance increased with the amount of polymerase and with the time of preincubation. Since the resistance to nuclease was not observed in the presence of rifampicin or by preincubation without UTP, both RNA chain initiation and elongation were considered to be essential for the manifestation of resistance. However, when DNase 1 was used as a probe, such a change in chromatin DNA was not detected.
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PMID:Transcription renders chromatin resistant to micrococcal nuclease digestion. 635 97

When rat liver nuclei were digested with nuclease, we found that the chromatin-bound RNA polymerase II was liberated as two distinct complexes, peak 1 and peak 2, which seemed to reflect different functional states in cell nuclei. We further examined their occurrence in nuclear digests of various tissues of rats and the following results were obtained. Upon digestion with micrococcal nuclease of nuclei from brain, spleen, testis and kidney, chromatin-bound RNA polymerase II was liberated as two distinct forms which sedimented differently in a sucrose density gradient. The sedimentation rate of peak 1 varied depending on the tissue nuclei examined. After high salt or RNase treatment of the nuclear digests, peak 1 from liver, brain, spleen and testis nuclei showed the same sedimentation rate as did kidney peak 1, the rate for which remained unchanged by these treatments. The results suggested that peak 1 complexes from various tissue nuclei had basically the same structural organization, and we confirmed this by electrophoretic studies on RNase-treated liver and kidney nuclear digests. Peak 2 from various tissue nuclei exhibited identical sedimentation rates. Thus, the chromatin-bound RNA polymerase II seems to exist commonly in two distinct states in cell nuclei of rats.
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PMID:Two species of chromatin-RNA polymerase II complex are commonly present in nuclei of various tissues of rats. 652 8

Controlled digestion of rat ventral prostate nuclei by careful adjustment of conditions of temperature, divalent cation concentration, ionic strength and micrococcal nuclease:DNA ratios yielded oligonucleosome fractions corresponding to less than 10% of the total genome which contain the majority of RNA polymerase B activity and androgen-receptor complexes of the nucleus. These parameters were affected acutely by androgen withdrawal and administration: furthermore, such manipulations affected the susceptibility to micrococcal nuclease release of prostate binding protein gene sequences. This transcriptionally-active androgen-influenced fraction was considered ideal for studies of interaction of chromatin components with androgen receptor protein. Androgen receptor was purified approximately 20 000-fold from rat prostate cytosol. The purified protein retained its ability to stimulate RNA polymerase B activity in prostate nuclei and chromatin fractions, and its properties of binding to chromatin and to DNA. However, although purified receptor protein showed tissue-specific binding to prostate chromatin and enhanced binding to fractions released by low nuclease digestion, no such specificity was indicated by binding to total DNA, DNA from specific fractions or cloned prostatic binding protein cDNA.
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PMID:Interaction of androgen receptors with chromatin and DNA. 653 19

A method is described for preparation of a fraction of chromatin enriched in transcribing regions from nuclei of mouse GR cells. This fraction, released by mild staphylococcal nuclease digestion of isolated nuclei, contains 2 to 10% of the DNA as polynucleosomal chromatin together with 50-70% of pulse-labelled RNA and about 90% of all template-engaged RNA polymerase B molecules, titrated with (3H)-alpha-amanitin. Hybridisation of DNA from this chromatin fraction to total nuclear RNA in excess shows that it is enriched in frequently-transcribed DNA sequences. A modification of the Miller technique, allowing the spreading of the active chromatin fraction for electron microscopy, has been developed. Examination of the spreads reveals that this chromatin fraction contains 20-100 nucleosome-long polynucleosomal chains bearing lateral RNP fibrils interpreted as nascent transcripts. The average length of the DNA fragments in the fraction is greater than that of average transcribed regions, suggesting that the transcribed regions are linked to flanking segments whose chromatin conformation probably contributes to the selective release of transcribing chromatin.
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PMID:Isolation and characterisation of a transcribing polynucleosomal chromatin fraction. 667 92

Chromatin fragments of the RNA polymerase II-transcriptional complex were purified from the micrococcal nuclease digest of rat liver nuclei in the presence of n-butyrate, a potent histone deacetylase inhibitor. Polyacrylamide gel electrophoretic analysis in Triton acid-urea revealed that the extent of histone acetylation of the complex did not differ markedly from that of the total chromatin.
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PMID:Transcribing chromatin is not preferentially enriched with acetylated histones. 687 81


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