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 ultraviolet sensitivity of Potorous tridactylus male kidney (PtK-2) cells is markedly increased by post irradiation treatment for 24 h with 5 microM emetine (which inhibits protein synthesis by acting on the 40S ribosomal subunit), or with 5 microM cycloheximide (which inhibits by interaction with the 60S subunit), or with the RNA polymerase II inhibitor 5,6-dichloro-1-beta-ribofuranosylbenzimidazole at 50 microM. All 3 treatments give the same sensitivity, while unirradiated cells are little affected. Shortening the time of treatment, or delaying application of the drugs decreases their effect on the same time schedule. Preiirradiation of cells, with no drug treatment in the following 8 h, diminishes the sensitivity to a subsequent irradiation with protein synthesis blocked afterwards. Photoreactivation immediately following such preirradiation eliminates its desensitizing effect. Inhibiting protein synthesis after irradiation also markedly reduces the frequency of UV-induced mutants in the surviving population. These facts suggest that gene expression in the period following irradiation facilitates recovery from radiation damage, with an increased probability of mutation, reminiscent of the "SOS response" in Escherichia coli.
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PMID:Radiation-induced recovery processes in cultured marsupial cells. 341 65

The X. laevis ribosomal DNA spacer contains duplicated RNA polymerase I "spacer promoters" and an array of repeated 60/81 bp promoter-related sequences. The latter have been shown to enhance transcription from a 40S preribosomal RNA promoter in cis. Here we present evidence that at least one spacer promoter is also necessary for efficient enhancement. Deletion of the spacer promoter sequences in a construct carrying only one such promoter reduces 40S RNA transcription to approximately 10% of wild type. This effect apparently is caused by inactivation of the spacer promoter, since mutants in which 4-177 bp of the spacer promoter and adjacent sequences are deleted are functionally equivalent. Spacer promoters and 60/80 bp arrays therefore probably act together to enhance 40S pre-RNA transcription in X. laevis.
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PMID:Spacer promoters are essential for efficient enhancement of X. laevis ribosomal transcription. 394 26

Previous data demonstrated that reovirus mRNA, synthesized in vitro with the particulate RNA transcriptase of reovirus cores, efficiently directs the synthesis of polypeptides in vitro. The present studies indicate that all of the three size classes of reovirus mRNA produced in vitro can form protein initiation complexes with rat liver [(36)S]Met-tRNA(F) and incubated 40S and 60S ribosomal subunits, which had been washed in 0.5 M KCl of mouse fibroblast L-929 cells. Mild prior treatment of the mRNA with HCHO was required to expose the initiator region. The initiation complex reacted quantitatively with puromycin to form a puromycin peptide, whose electrophoretic properties were identical to methionyl-puromycin formed in response to poly(A,G,U) or the initiator codon AUG. The complex was relatively stable and specific for [(35)S]Met-tRNA(F); rat liver [(35)S]Met-tRNA(M) was unreactive unless the supernatant factors EF T(1) and EF T(2) were also present. However, the addition of fusidic acid, at a concentration that did not affect complex formation with [(35)S]Met-tRNA(F), completely inhibited Met-tRNA(M) utilization. Exogenous ribosomal factors and GTP were not required unless the separated 40S and 60S subunits were further treated with 1 M KCl. The data suggest that reovirus mRNA contains AUG initiator codons that form a complex with Met-tRNA(F) at a puromycin-reactive site on ribosomes.
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PMID:Formation of a mammalian initiation complex with reovirus messenger RNA, methionyl-tRNA F , and ribosomal subunits. 450 35

A two-step procedure has been developed for the formation of RNA polymerase II transcription initiation and elongation complexes. Initiation complexes are rapidly formed in HeLa cell-free extract supplemented with a DNA template containing the adenovirus 2 major late promoter and ATP. Assembly of transcription components required for correct initiation is absolutely dependent on specific eukaryotic promoter sequences. Sarkosyl-sensitive transcription initiation complexes are rapidly converted to Sarkosyl-resistant elongation complexes when supplemented with the remaining nucleoside triphosphates. The 60S initiation complex can be extensively purified by glycerol gradient centrifugation and is easily separated from free RNA polymerase II and free DNA template. Recovery of this stable complex is greater than 90%. Specific transcription cannot be detected if the DNA template is subsequently added to gradient fractions containing HeLa cell-free extract components alone. This suggests that the DNA templates promote the specific assembly of RNA polymerase II and transcription factors required for accurate initiation. Since conversion of purified initiation complexes to elongation complexes can occur without additional HeLa cell components, the presence of transcription components required for initiation and elongation in a single complex is indicated.
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PMID:Isolation of an active transcription initiation complex from HeLa cell-free extract. 659 95

We have isolated germinal vesicles from previtellogenic Xenopus oocytes on a scale suitable for biochemical studies. The organelles were active in RNA synthesis, mainly mediated by endogenous RNA polymerase I. Nascent transcripts were 5-18S in size and complementary to DNA sequences present at intermediate reiteration frequency (several hundred copies per haploid genome). Hybridization to cloned 5S and 40S ribosomal genes demonstrated that the in vitro transcripts contained the latter, but not the former sequences in detectable amounts. The germinal vesicles also exhibited an ability to take up and retain UTP and a UTP-binding activity, probably protein(s), was extractable from the organelles with 0.35 M NaCl.
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PMID:Transcriptional activity in previtellogenic oocyte germinal vesicles from Xenopus laevis. 685 26

Transcription of a cloned Xenopus laevis ribosomal DNA (rDNA) fragment, microinjected into Xenopus oocytes, is initiated at the in vivo 40S pre-ribosomal RNA (pre-rRNA) site (+/- 2 bp) by RNA polymerase I. An X. laevis RNA polymerase I promoter has been mapped by studying the transcription of in vitro rDNA mutants in the oocyte system. The active promoter lies within the DNA segment beginning 145 bp upstream, and most probably ending 16 bp downstream, from the 40S pre-rRNA initiation site (-145 bp to +16 bp). Furthermore, active promoter elements lie more than 35 bp upstream from the initiation site (-35 bp). The X. laevis RNA polymerase I promoter therefore lies mainly upstream from the 40S pre-rRNA initiation site. Independent deletion of three adjacent rDNA segments lying between -61 and +16 bp reduces promoter activity by a factor of more than 16. The central of these "null" deletions removes an oligo(T)6 motif at -27 bp that is in an analogous position to the Goldberg-Hogness (TATA) box of RNA polymerase II promoters.
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PMID:Transcription of cloned Xenopus laevis ribosomal DNA microinjected into Xenopus oocytes, and the identification of an RNA polymerase I promoter. 713 16

The ultrastructure of Xenopus laevis (X.l.) 40S pre-rRNA transcription termination sites was investigated by electron microscopy. Amplified nucleolar chromatin was rapidly dispersed and processed for chromatin spread preparations. This type of preparation revealed that many rDNA termination sites appeared as 50 nm long segments of chromatin axis covered by a complex of three closely spaced RNA polymerase particles. Particle (Ptl) was characterized by the association with the terminal full-length pre-rRNP fibril; particles (Pt2) and (Pt3) are located downstream from (Ptl) and appear to be devoid of transcript fibrils. This particular structural arrangement as well as sequence homology analyses of 3' adjacent spacer rDNA segments indicate that transcript release and dissociation of polymerase particles are not necessarily coupled and termination of X.l. rDNA transcription may occur as a two step process. The structural data are correlated with homologies of DNA sequences at Xenopus rDNA transcription termination regions and are discussed with respect to sequence data of 3' termination sites of rRNA genes from other species.
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PMID:Chromatin structure of Xenopus rDNA transcription termination sites. Evidence for a two-step process of transcription termination. 715 44

We have developed a system for mutational analysis of Saccharomyces cerevisiae ribosomal RNA in vivo in which yeast cells can be made completely dependent on mutant rRNA and ribosomes by a simple switch in carbon source. The system is based on a yeast strain defective in RNA polymerase I (Pol I) transcription [Nogi et al. (1991) Proc. Natl. Acad. Sci. USA 88, 3962-3966]. This normally inviable strain was rescued by integration of multiple copies of the complete 37S pre-rRNA operon under control of the inducible, Pol II-transcribed GAL7 promoter into the rDNA repeat on chromosome XII. The resulting YJV100 strain can only grow on medium containing galactose as the carbon source. A second, episomal vector was constructed in which the rDNA unit was placed under control of the constitutive PGK1 promoter. YJV100 cells transformed with this vector are now also able to grow on glucose-based medium making the cells completely dependent on plasmid-encoded rRNA. We show that the Pol II-transcribed pre-rRNA is processed and assembled similarly to authentic Pol I-synthesised pre-rRNA, making this 'in vivo Pol II system' suitable for the detailed analysis of rRNA mutations, even highly deleterious ones, affecting ribosome biogenesis or function. A clear demonstration of this is our finding that an insertion into variable region V8 in 17S rRNA, previously judged to be neutral with respect to processing of 17S rRNA, its assembly into 40S subunits and the polysomal distribution of these subunits [Musters et al. (1989), Mol. Cell. Biol. 9, 551-559], is in fact a lethal mutation.
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PMID:Development and application of an in vivo system to study yeast ribosomal RNA biogenesis and function. 773 24

Eukaryotic translation initiation factor 5, eIF-5, has been purified to apparent electrophoretic homogeneity from overproducing Escherichia coli cells expressing the cDNA of the initiation factor under the control of the T7 promoter-T7 RNA polymerase system. Purified recombinant eIF-5 mimics natural eIF-5 isolated from mammalian cells in size, in specific activity, in its ability to catalyze the hydrolysis of GTP bound to the 40S initiation complex, and in the subsequent joining with 60S ribosomal subunits to form the 80S initiation complex. Further characterization of eIF-5 demonstrates that eIF-5 specifically associates with eIF-2, forming an eIF-2.eIF-5 complex. The protein complex sediments in glycerol gradients with an apparent M(r) of 160,000, suggesting that the two proteins associate in a 1:1 stoichiometry. The association between the two initiation factors is highly specific. Addition of 32P-labeled eIF-5 to a partially purified rabbit reticulocyte initiation factor preparation that contained, in addition to eIF-2 and eIF-5, other initiation factors and many other proteins resulted in the specific binding of labeled eIF-5 only to eIF-2, forming a 160-kDa protein complex. In agreement with these observations, we found that in crude initiation factor preparations derived from rabbit reticulocyte lysates, eIF-5 was present as an eIF-2.eIF-5 complex. The significance of eIF-2.eIF-5 complex formation in the overall mechanism of GTP hydrolysis in protein synthesis initiation is discussed.
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PMID:Purification and characterization of bacterially expressed mammalian translation initiation factor 5 (eIF-5): demonstration that eIF-5 forms a specific complex with eIF-2. 816 39

The interaction of the ribosomal transcription factor xUBF with the RNA polymerase I core promoter of Xenopus laevis has been studied both at the DNA and protein levels. It is shown that a single xUBF-DNA complex forms over the 40S initiation site (+1) and involves at least the DNA sequences between -20 and +60 bp. DNA sequences upstream of +10 and downstream of +18 are each sufficient to direct complex formation independently. HMG box 1 of xUBF independently recognizes the sequences -20 to -1 and +1 to +22 and the addition of the N-terminal dimerization domain to HMG box 1 stabilizes its interaction with these sequences approximately 10-fold. HMG boxes 2/3 interact with the DNA downstream of +22 and can independently position xUBF across the initiation site. The C-terminal segment of xUBF, HMG boxes 4, 5 or the acidic domain, directly or indirectly interact with HMG box 1, making the core promoter sequences between -11 and -15 hypersensitive to DNase. This interaction also requires the DNA sequences between +17 and +32, i.e. the HMG box 2/3 binding site. The data suggest extensive folding of the core promoter within the xUBF complex.
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PMID:Recognition of the Xenopus ribosomal core promoter by the transcription factor xUBF involves multiple HMG box domains and leads to an xUBF interdomain interaction. 844 Feb 41


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