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)

Escherichia coli RNA Polymerase is a multi-subunit enzyme that catalyzes RNA synthesis, using DNA as a template. The sigma subunit of this enzyme plays an important role in the recognition of promoter sites on DNA. Using DNase I footprinting, we have found that in the absence of the other subunits, sigma binds specifically to the bacteriophage lambda PR promoter DNA sequence. In the presence of the sigma subunit alone, a protective footprint encompassing the region between residue positions -41 and +17 was observed (where +1 is the transcription start site). The holoenzyme gave a footprint covering the same region. Thus not only does the sigma subunit interact with the DNA promoter site in the absence of the other components of RNA polymerase, but also the footprint of sigma is indistinguishable from that of the holoenzyme.
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PMID:Footprint of the sigma protein. 252 2

We have investigated the factors that permit a gene normally transcribed by RNA polymerase II to be transcribed by RNA polymerase III. It was shown previously that the human c-myc gene could be transcribed in vitro and in Xenopus oocytes by both alpha-amanitin-sensitive and alpha-amanitin-resistant polymerases, probably corresponding to polymerase II and polymerase III. We confirmed this observation in microinjected oocytes and showed that the alpha-amanitin-resistant transcription of c-myc was competed by known polymerase III genes. Polymerase III transcription of c-myc was very inefficient compared to other polymerase III genes, however, and was observed only when large amounts of template DNA were injected. At lower DNA concentrations the gene was transcribed, exclusively by polymerase II. In contrast, the adenovirus major late promoter was not transcribed by polymerase III. The 5' ends of polymerase III RNAs were almost indistinguishable from those of polymerase II RNAs initiating at the P1 and P2 promoters of the human and mouse c-myc genes. Furthermore, point mutations in the TATA box of the human P2 promoter greatly reduced polymerase III activity. At this promoter, therefore, polymerase II and polymerase III recognize a common element, the TATA box, which probably plays an important role in specifying the start site of transcription for both polymerases. We suggest that the highly accurate though inefficient mimicry of polymerase II by polymerase III at the c-myc promoters reflects the common evolutionary origin of these two enzymes.
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PMID:Accurate, TATA box-dependent polymerase III transcription from promoters of the c-myc gene in injected Xenopus oocytes. 279 59

The in vitro amplification of biologically important nucleic acids has proceeded principally by a strategy of DNA replication. Polymerase chain reaction was the first such protocol to achieve this goal. In this report, a transcription-based amplification system (TAS) is described. Each cycle of the TAS is composed of two steps. The first is a cDNA synthesis step that produces one copy of a double-stranded DNA template for each copy of RNA or DNA target nucleic acid. During the course of this cDNA synthesis step, a sequence recognized by a DNA-dependent RNA polymerase is inserted into the cDNA copy of the target sequence to be amplified. The second step is the amplification of the target sequence by the transcription of the cDNA template into multiple copies of RNA. This procedure has been applied to the detection of human immunodeficiency virus type 1 (HIV-1)-infected cells. After four cycles of TAS, the amplification of the vif region of the HIV-1 RNA genome was measured to be, on the average, 38- to 47-fold per cycle, resulting in a 2-5 x 10(6)-fold increase in the copy number of the original target sequence. This amplification by the TAS protocol allows the detection of fewer than one HIV-1-infected CEM cell in a population of 10(6) uninfected CEM cells. Detection of the TAS-generated RNA from HIV-1-infected cells can easily be accomplished by means of a bead-based sandwich hybridization protocol, which provides additional specificity for the identification of the amplified HIV-1-specific sequence.
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PMID:Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format. 291 66

The poliovirus RNA polymerase, 3Dpol, was used to synthesize RNA in vitro in the presence of a host factor preparation from uninfected HeLa cells and poliovirion RNA as the template. The transcription products included molecules approximately twice the length of the template, apparently resulting from hairpin formation and template-directed elongation, as previously reported (D. C. Young, D. M. Tuschall, and J. B. Flanegan, J. Virol. 54:256-264, 1985). Other polyadenylated template RNAs also yielded products that were twice the length of the template. The polarity of the products synthesized from plus-strand poliovirus RNA template was analyzed by Southern blotting using labeled product RNA to probe single-stranded poliovirus DNAs cloned into M13 vectors. The results demonstrated that host factor-mediated polymerase products contain newly synthesized plus-strand sequences as well as the expected minus-strand sequences. Polymerase products primed with oligo(U) were all of minus-strand polarity.
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PMID:Synthesis of plus- and minus-strand RNA from poliovirion RNA template in vitro. 300 87

RNA polymerase I binding to the eukaryotic ribosomal RNA gene promoter-transcription initiation factor (TIF) complex was examined by in vitro transcription and footprinting of a series of spacer mutants. Polymerase binds efficiently to the TIF-promoter complex independently of the DNA sequence in the polymerase interaction region and initiates transcription a fixed distance downstream of the TIF binding site on AT-rich templates. Methidiumpropyl-EDTA.FE(II) footprinting confirms minimal contacts between polymerase and DNA. We infer that polymerase is directed to the promoter by a DNA sequence-independent mechanism, solely by protein-protein contacts with TIF. An initiation step subsequent to binding requires special sequence characteristics in the transcription start site region.
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PMID:Eukaryotic RNA polymerase I promoter binding is directed by protein contacts with transcription initiation factor and is DNA sequence-independent. 311 36

We have isolated, characterized and substantially purified two distinct RNA polymerase activities from the flagellate protozoan parasite Trypanosoma brucei. RNA polymerases from this organism were resolved poorly on DEAE-Sephadex, but could be separated with CM-Sephadex. One form was totally resistant to alpha-amanitin, whereas the second was 50% inhibited by 10-20 micrograms of the drug/ml. The enzymes had different salt optima, but both were of high Mr (greater than 480,000) and demonstrated the template preference: poly[d(A-T)] greater than denatured DNA greater than native DNA, and both were more active with Mn2+ than with Mg2+. The amanitin-resistant enzyme, polymerase R, was partially purified by chromatography on CM-Sephadex, DEAE-Sephadex and heparin-Sepharose. This enzyme was very labile, and activity yields were around 9%; after purification, one or two protein bands could be discerned after electrophoresis under non-denaturing conditions, but about 20 polypeptides were resolved on denaturing gels, including a major component (not thought to be part of the enzyme) of Mr 65,000. Polymerase S, sensitive to low alpha-amanitin concentrations, was more extensively purified, with an 18% recovery, and yielded a single major band with two minor ones after native gel electrophoresis. Analysis under denaturing conditions permitted a possible subunit structure for this enzyme to be ascribed.
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PMID:Demonstration of RNA polymerase multiplicity in Trypanosoma brucei. Characterization and purification of alpha-amanitin-resistant and -sensitive enzymes. 359 14

Forms of RNA polymerase I prepared from growing or encysted Acanthamoeba are equal in the ability to transcribe poly(dl:dC). Polymerase from cysts, whose rRNA genes are transcriptionally inactive, is unable to utilize the rDNA promoter in vitro, whereas the transcription initiation factor from cysts is fully able to bind the promoter and direct transcription. Footprinting shows that polymerase from cysts is functionally inactive because of its inability to bind to the promoter. The polymerase footprint moves downstream the appropriate number of base pairs upon various nucleotide additions, without affecting the factor footprint. These results support our hypothesis that rRNA synthesis in eukaryotes is regulated by polymerase I modification and not by alterations to additional DNA-binding proteins.
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PMID:Regulation of eukaryotic ribosomal RNA transcription by RNA polymerase modification. 376 60

We have measured the sedimentation coefficients (s) of different DNA molecules of a few thousand base bairs in the presence of increasing amounts of E. coli RNA polymerase under conditions where tight binding complexes are formed. The measured s does not increase linearly with n(n=RNA Polymerase/DNA molar ratio); the s vs n plot can be decomposed into two parts; first the increase in s is small until n reaches a value n0 approximately equal to the number of strong promoters of the DNA molecule under consideration, then when n greater than n0 the slope of s(n) is much higher. The observations are in agreement with a model which postulates that strong specific polymerase binding leads to an increase in frictional coefficient of the RNA Polymerase-DNA complex, while non specific(or less specific)RNAP binding leads to a contraction of the RNA Polymerase-DNA complexes.
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PMID:Specific and non specific Escherichia coli ribonucleic acid polymerase DNA complexes are not hydrodynamically equivalent in analytical band sedimentation. 389 25

A procedure for the separation and purification of DNA-dependent RNA polymerases [EC 2.7.7.6] from macronuclei of Tetrahymena pyriformis is described. We have used it to isolate and characterize the class I enzyme. RNA polymerase I was identified by its resistance against alpha-amanitin and its location in nucleoli. The purified enzyme consists of at least 12 major subunits with approximate molecular weights of 180,000, 118,000, 37,500, 36,000, 29,000, 27,500, 20,000, 18,500, 15,600, 14,500, 13,500, and 12,600. Chromatography on DEAE-Sephadex separated two forms of RNA polymerase I which differed in the presence of an additional polypeptide of 25 kDa. Independently of this polypeptide, the enzyme was found to segregate on DNA cellulose into a binding and a non-binding fraction. This type of heterogeneity was found to be unrelated to differences in molar ratios or molecular weights of the enzyme subunits. The catalytic properties of all enzyme subfractions were very similar and complied with the general characteristics of RNA polymerase I [cf. Roeder, R.G. (1976) in RNA Polymerase (Losick, R. & Chamberlin, M., eds.) pp. 285-329, Cold Spring Harbor Publ. Co., New York].
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PMID:Isolation and characterization of DNA-dependent RNA polymerase I of Tetrahymena pyriformis. 393 46

RNA polymerase activities in intact HeLa cell nuclei have been examined and compared to activities investigated in previous studies of the purified enzymes. The RNA synthesized by the mammalian polymerases while still in nuclei is identified. The polymerases are tentatively identified by location, sensitivity to alpha-amanitin, and response to manganese and altered ionic strength. Polymerase I is located in the nucleolus and labels partially complete precursor molecules of ribosomal RNA. Polymerases II and III are in the nucleoplasm and both label giant nuclear heterogeneous RNA.
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PMID:Products of RNA polymerases in HeLa cell nuclei. 410 11


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