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 rate of dissociation of actinomycin D from DNA was measured by sodium dodecyl sulphate (SDS) sequestration (37 degrees) from calf thymus DNA and a 24 base pair (bp) synthetic DNA containing one high affinity AGCT site for the drug. The time constants were 276 and 142 sec, respectively, and suggest a stabilising effect though positive cooperativity in heterogenous DNA, or from specific neighbouring sequences. The time constant for dissociation of actinomycin D from the AGCT site was 2900 sec as measured by an in vitro transcription assay at 37 degrees, and suggests that, under conditions of active transcription of the DNA, the drug-DNA complex has additional stabilising contributions, possibly by a cage effect from RNA polymerase, or by additional drug-RNA polymerase contacts.
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PMID:Drug-DNA dissociation kinetics. In vitro transcription and sodium dodecyl sulphate sequestration. 291 17

Native chromatin aggregates under isotonic conditions so it is generally studied using higher or lower salt concentrations. This has led to different interpretations of how transcription might occur. Studies using hypertonically-isolated preparations suggest that DNA functions in close association with a skeletal nuclear substructure, the matrix or cage, but such a structure is not usually seen under hypotonic conditions (e.g., in 'Miller-spreads'). Using a novel method for preparing chromatin under isotonic conditions we have investigated the site of transcription. We find that all three constituents of the transcription complex, nascent transcripts, active RNA polymerase and genes being transcribed are all closely associated with some structure too large to be electroeluted from the nucleus. Hypotonic treatment partly disrupts this association. We suggest a model for transcription that involves the participation of a nucleoskeleton at the active site and reconcile the contradictory results obtained using different salt concentrations.
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PMID:Transcription occurs at a nucleoskeleton. 299 Sep 13

Frequently, in nature, defective promoters can be resurrected by activator proteins in response to cellular demands. The activators bind to nearby DNA sites for action. Various protein-protein and DNA-protein contacts involving activators, RNA polymerase, and different segments of DNA in and around a defective promoter form a DNA-multiprotein complex (cage) which enhances transcription.
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PMID:Promoter resurrection by activators--a minireview. 840 30

Pariacoto virus (PaV) is a nodavirus that was recently isolated in Peru from the Southern armyworm, Spodoptera eridania. Virus particles are non enveloped and about 30 nm in diameter and have T=3 icosahedral symmetry. The 3.0-A crystal structure shows that about 35% of the genomic RNA is icosahedrally ordered, with the RNA forming a dodecahedral cage of 25-nucleotide (nt) duplexes that underlie the inner surface of the capsid. The PaV genome comprises two single-stranded, positive-sense RNAs: RNA1 (3,011 nt), which encodes the 108-kDa catalytic subunit of the RNA-dependent RNA polymerase, and RNA2 (1,311 nt), which encodes the 43-kDa capsid protein precursor alpha. In order to apply molecular genetics to the structure and assembly of PaV, we identified susceptible cell lines and developed a reverse genetic system for this virus. Cell lines that were susceptible to infection by PaV included those from Spodoptera exigua, Helicoverpa zea and Aedes albopictus, whereas cells from Drosophila melanogaster and Spodoptera frugiperda were refractory to infection. To recover virus from molecular clones, full-length cDNAs of PaV RNAs 1 and 2 were cotranscribed by T7 RNA polymerase in baby hamster kidney cells that expressed T7 RNA polymerase. Lysates of these cells were infectious both for cultured cells from Helicoverpa zea (corn earworm) and for larvae of Galleria mellonella (greater wax moth). The combination of infectious cDNA clones, cell culture infectivity, and the ability to produce milligram amounts of virus allows the application of DNA-based genetic methods to the study of PaV structure and assembly.
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PMID:Recovery of infectious pariacoto virus from cDNA clones and identification of susceptible cell lines. 1171 13

Reverse transcriptase-polymerase chain reaction (RT-PCR) assays have proved useful for the detection of mouse hepatitis virus (MHV) and rat coronavirus (RCV) in acutely infected animals and contaminated biomaterials. Fluorogenic nuclease RT-PCR assays combine RT-PCR with an internal fluorogenic hybridization probe, thereby eliminating post-PCR processing and potentially enhancing specificity. Consequently, a fluorogenic nuclease RT-PCR assay specific for rodent coronaviruses was developed. Primer and probe sequences were selected from the viral genome segment that encodes the membrane (M) protein that is highly conserved among rodent coronaviruses. Use of the fluorogenic nuclease RT-PCR detected all strains of MHV and RCV that were evaluated, but did not detect other RNA viruses that naturally infect rodents. Use of the assay detected as little as two femtograms of in vitro transcribed RNA generated from cloned amplicon, and when compared directly with mouse antibody production tests, had similar sensitivity at detecting MHV-A59 in infected cell culture lysates. Finally, use of the assay detected coronavirus RNA in tissues, cage swipes, and feces obtained from mice experimentally infected with MHV, and in tissues and cage swipes obtained from rats naturally infected with RCV. These results indicate that the fluorogenic nuclease RT-PCR assay should provide a potentially high-throughput, PCR-based method to detect rodent coronaviruses in infected rodents and contaminated biological materials.
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PMID:Detection of rodent coronaviruses by use of fluorogenic reverse transcriptase-polymerase chain reaction analysis. 1202 89

Sendai virus may induce acute respiratory tract disease in laboratory mice and is a common contaminant of biological materials. Pneumonia virus of mice (PVM) also infects the respiratory tract and, like Sendai virus, may induce a persistent wasting disease syndrome in immunodeficient mice. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays have proven useful for detection of Sendai virus and PVM immunodeficient animals and contaminated biomaterials. Fluorogenic nuclease RT-PCR assays (fnRT-PCR) combine RT-PCR with an internal fluorogenic hybridization probe, thereby potentially enhancing specificity and eliminating post-PCR processing. Therefore, fnRT-PCR assays specific for Sendai virus and PVM were developed by targeting primer andprobe sequences to unique regions of the Sendai virus nucleocapsid (NP) gene and the PVM attachment (G) gene, respectively. The Sendai virus and PVM fnRT-PCR assays detected only Sendai virusand PVM , respectively. Neither assay detected other viruses of the family Paramyxoviridae or other RNA viruses that naturally infect rodents. The fnRT-PCR assays detected as little as 10 fg of Sendai virus RNA and one picogram of PVM RNA, respectively, andthe Sendai virus fnRT-PCR assay had comparable sensitivity when directly compared with the mouse antibody production test. The fnRT-PCR assays were also able to detect viral RNA in respiratory tract tissues and cage swipe specimens collected from experimentally inoculated C.B-17 severe combined immunodeficient mice, but did not detect viral RNA in age- and strain-matched mock-infected mice. In conclusion, these fnRT-PCR assays offer potentially high-throughput diagnostic assays to detect Sendai virus and PVM in immunodeficient mice, and to detect Sendai virus in contaminated biological materials.
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PMID:Detection of sendai virus and pneumonia virus of mice by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction analysis. 1278 51

EBNA 2 is one of only five viral genes essential for the infection and immortalization of human B cells by the cancer-associated virus Epstein-Barr virus (EBV). EBNA 2 activates cellular and viral transcription and associates with components of the basal transcription apparatus and a number of coactivators. We provide the first evidence to show that the mechanism of transcriptional activation by EBNA 2 also involves phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (pol II). We found that transcriptional activation by EBNA 2 was inhibited by a dominant-negative mutant of the pol II CTD kinase, CDK9, and by low concentrations of the CDK9 inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Moreover, using chromatin immunoprecipitation assays we demonstrated that EBNA 2 stimulates both pol II recruitment and pol II phosphorylation on serine 5 of the CTD in vivo. These results identify a new step in the transcription cycle that is subject to regulation by a key EBV-encoded transcription factor and highlight CDK9 inhibitors as potential anti-EBV agents.
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PMID:EBV EBNA 2 stimulates CDK9-dependent transcription and RNA polymerase II phosphorylation on serine 5. 1631 42

Short hairpin RNAs (shRNAs) transcribed by RNA polymerase III promoters can trigger sequence-selective gene silencing in mammalian cells. By virtue of their excellent function in knocking down expression of cancer-associated genes, shRNAs could be used as new therapeutic agents for cancer. As overexpression of Ki67 in renal cancer has been correlated to a more aggressive tumor phenotype, inhibition of Ki67 protein expression by means of shRNAs seems to be a promising approach for the therapy of renal cancer. In this study, we constructed an expression plasmid encoding shRNAs against the Ki67 gene, named pSilencerKi67, and transfected it into human renal carcinoma cells. The pSilencerKi67 was shown to significantly knock down the expression of the Ki67 gene in human renal carcinoma cells, resulting in inhibiting proliferation and inducing apoptotic cell death that can be maintained for at least 6 d. These findings offer the promise of using vector-based shRNAs against Ki67 in renal cancer gene therapy.
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PMID:Treatment with vector-expressed small hairpin RNAs against Ki67 RNA-induced cell growth inhibition and apoptosis in human renal carcinoma cells. 1660 65

Double chromodomains occur in CHD proteins, which are ATP-dependent chromatin remodeling factors implicated in RNA polymerase II transcription regulation. Biochemical studies suggest important differences in the histone H3 tail binding of different CHD chromodomains. In human and Drosophila, CHD1 double chromodomains bind lysine 4-methylated histone H3 tail, which is a hallmark of transcriptionally active chromatin in all eukaryotes. Here, we present the crystal structure of the yeast CHD1 double chromodomains, and pinpoint their differences with that of the human CHD1 double chromodomains. The most conserved residues in these double chromodomains are the two chromoboxes that orient adjacently. Only a subset of CHD chromoboxes can form an aromatic cage for methyllysine binding, and methyllysine binding requires correctly oriented inserts. These factors preclude yeast CHD1 double chromodomains from interacting with the histone H3 tail. Despite great sequence similarity between the human CHD1 and CHD2 chromodomains, variation within an insert likely prevents CHD2 double chromodomains from binding lysine 4-methylated histone H3 tail as efficiently as in CHD1. By using the available structural and biochemical data we highlight the evolutionary specialization of CHD double chromodomains, and provide insights about their targeting capacities.
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PMID:Molecular implications of evolutionary differences in CHD double chromodomains. 1743 64

The EBNA 2 (Epstein-Barr nuclear antigen 2) transcription factor is essential for B-cell transformation by the cancer-associated EBV (Epstein-Barr virus) and for the continuous proliferation of infected cells. EBNA 2 activates transcription from the viral Cp (C promoter) during infection to generate the 120 kb transcript that encodes all nuclear antigens required for immortalization by EBV. EBNA 2 contains an acidic activation domain and can interact with a number of general transcription factors and co-activators. It is now becoming clear, however, that the regulation of transcription elongation in addition to initiation by EBNA 2, at least in part through CDK9 (cyclin-dependent kinase 9)-dependent phosphorylation of the RNA polymerase C-terminal domain, is likely to play a crucial role in the mechanism of action of this key viral protein.
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PMID:Regulation of transcription by the Epstein-Barr virus nuclear antigen EBNA 2. 1863 Nov 29


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