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)

DRB triphosphate inhibits activity of isolated RNA polymerase B, and, to a lesser extent, that of polymerase A. The same holds true for transcription in isolated nuclei. It does not act as an initiation inhibitor. In all cases, high concentrations of DRB triphosphate are required. Cells do not phosphorylate DRB to a measurable extent. hn RNA resistant to DRB is initiated with both ATP and GTP in the presence of the drug. These experiments render the hypothesis unlikely that DRB triphosphate in the cell specifically interferes with the initiation reaction of polymerase B.
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PMID:Inhibition of mammalian RNA polymerase by 5,6-dichlororibofuranosylbenzimidazole (DRB) and DRB triphosphate. 70 59

We have studied the ultrastructure of the Balbiani ring genes in Chironomus tentans during treatment with the RNA synthesis inhibitor DRB (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole). This nucleoside analogue blocks transcription at or near the initiation site but does not interfere with the elongation and termination processes. In the ordinary active state the Balbiani ring genes display a 5 nm chromosome fiber, carrying densely distributed, growing ribonucleoprotein particles (Andersson et al., 1980). When the transcriptional activity declines, a 10 nm fiber can be observed between sparsely distributed RNA polymerases. Furthermore, after passage of the last RNA polymerase the 10 nm fiber can be seen as well as its gradual packing into a 25 nm thick fiber. Thus, the active chromosome fiber is rapidly packed into higher order structures when the fiber is not directly involved in transcription. The formation of the thick fiber does not require that the gene along its entire length is devoid of active RNA polymerases. The thick fiber can again be mobilized for transcription, since in reversion experiments the BR genes appear as ordinary active genes with an extended nucleofilament and densely packed nascent transcription products. The dynamic behaviour of the chromosome fiber during transcription is discussed as well as the packing and unpacking of a gene into higher order structures.
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PMID:Rapid reformation of the thick chromosome fiber upon completion of RNA synthesis at the Balbiani ring genes in Chironomus tentans. 618 41

To determine the role of DRB in transcription, we isolated a resistant (DRBR) HeLa cell mutant. After mutagenesis with N-methyl-N'-nitro-nitrosoguanidine, cell colonies able to grow at 20 micrograms DRB/ml (63 microM) were selected. One of these colonies, DRBR-1, was stable and able to grow at concentrations of DRB three to five times higher than tolerated by normal HeLa cells. The DNA of DRBR-1 was able to confer resistance to DRB to other HeLa cells by transfection. Uridine uptake was reduced by DRB to a similar extent in both wild-type and mutant cells. In contrast, transcription in the mutant cells, as measured by [3H]uridine incorporation into RNA in short pulses, was resistant to DRB. Cell-free extracts prepared from DRBR-1 cells are able to transcribe the epsilon-globin or the adenovirus 2 major late promoter genes at DRB concentrations that eliminate the transcriptional activity of HeLa cell extracts. Thus the transcriptional machinery of the mutant is altered. The presence of both DRB-resistant and DRB-sensitive transcriptional activities in extracts from DRBR-1 cells, grown in the presence of the drug, suggests constitutive expression of this cellular component. Efficient somatic cell hybridization with an alpha-amanitin-resistant RNA polymerase II mouse mutant indicates cross-complementation in vivo. This DRBR mutant provides a useful tool for the biochemical analysis of the mechanism of action of DRB on transcription. It also serves as a genetic handle for selection of the gene responsible for DRB resistance.
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PMID:Mechanism of action of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. II. A resistant human cell mutant with an altered transcriptional machinery. 618 48

The human immunodeficiency virus 1 (HIV-1) Rev transactivator protein plays a critical role in the regulation of expression of structural proteins by controlling the pathway of mRNA transport. The Rev protein is located predominantly in the nucleoli of HIV-1 infected or Rev-expressing cells. Previous studies demonstrated that the Rev protein forms a specific complex in vitro with protein B23 which is suggested to be a nucleolar receptor and/or carrier for the Rev protein. To study the role of the nucleolus and nucleolar proteins in Rev function, transfected COS-7 or transformed CMT3 cells expressing the Rev protein were examined for subcellular locations of Rev and other proteins using indirect immunofluorescence and immunoelectron microscopy. One day after transfection the Rev protein was found in most cells only in the nucleolar dense fibrillar and granular components where it colocalized with protein B23. These were designated class 1 cells. In a second class of cells Rev and B23 accumulated in the nucleoplasm as well as in nucleoli. Treatment of class 1 cells with actinomycin D (AMD) under conditions that blocked only RNA polymerase I transcription caused Rev to completely redistribute from nucleoli to the cytoplasm. Simultaneously, protein B23 was partially released from nucleoli, mostly into the nucleoplasm, with detectable amounts in the cytoplasm. In cells recovering from AMD treatment in the presence of cycloheximide Rev and B23 showed coincident relocation to nucleoli. Class 2 cells were resistant to AMD-induced Rev redistribution. Selective inhibition of RNA polymerase II transcription by alpha-amanitin or by DRB did not cause Rev to be released into the cytoplasm suggesting that active preribosomal RNA transcription is required for the nucleolar location of Rev. However, treatment with either of the latter two drugs at higher doses and for longer times caused partial disruption of nucleoli accompanied by translocation of the Rev protein to the cytoplasm. These results suggest that the nucleolar location of Rev depends on continuous preribosomal RNA transcription and a substantially intact nucleolar structure.
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PMID:The roles of nucleolar structure and function in the subcellular location of the HIV-1 Rev protein. 759 22

We are investigating the roles of RNA synthesis, chromatin structure and nuclear matrix organization in establishing and maintaining transcription domains, using mitogen stimulated lymphocytes as a model system. In a continuing study, the effects of the RNA polymerase inhibitor DRB and of its removal on nuclear organization have been examined by EM cytochemistry and by immunofluorescence labelling of the nuclear matrix PI1, Sm and nucleolar fibrillarin antigens. Chromatin, interchromatin granules and nucleoli were extensively restructured after DRB, as were matrix antigens. According to cytochemical staining properties, the conformation of DRB-induced condensed chromatin resembled that in partially stimulated lymphocytes. The nucleoplasmic fibrogranular RNP network appeared little altered, but the fibrillar proteinaceous interchromatinic regions, interpreted as representing the nuclear matrix in situ, were more affected. After removal of DRB, nuclei recovered the organization and transcriptional activity of controls within 8 h. These results suggest that the matrix subtending transcription domains remains stable when transcription is arrested, even though the chromatin and individual RNP components of the domains are disorganized. The data further indicate that absence of transcription is not solely accountable for the highly aggregated state of the chromatin in resting lymphocytes.
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PMID:Reversible disassembly of transcription domains in lymphocyte nuclei during inhibition of RNA synthesis by DRB. 769 22

Retinoids and transforming growth factor-beta 1 (TGF-beta 1) reduce the transcriptional activation of matrix metalloproteinases (MMPs) and increase the expression of the specific tissue inhibitor of MMPs (TIMP-1) in fibroblasts. In contrast, all-trans-retinoic acid (retinoic acid) increases MMP expression in osteoblasts. Therefore, the mechanistic aspects of TIMP-1 regulation by retinoic acid in primary cultures of rat calvarial bone cell populations were studied and compared with those of TGF-beta 1 to determine if modulation of TIMP-1 would augment MMP expression. Retinoic acid was found to reduce TIMP-1 mRNA levels after 24 and 72 hr of culture by up to 60% in a dose-dependent manner. Maximal inhibition occurred at 10(-6) M retinoic acid with half maximal repression at approximately 5 x 10(-8) M. To determine the half life of TIMP-1 mRNA, the specific RNA polymerase II inhibitor DRB was added to cultures and the chase RNA analyzed by slot blots. TIMP-1 mRNA had a half life of approximately 14 hr and this was unaltered by retinoic acid treatment, suggesting that retinoic acid exerts its effects on TIMP-1 transcriptionally. When retinoic acid was added to cycloheximide-treated cultures TIMP-1 mRNA levels were reduced at 5 hr compared with controls. This showed that ongoing protein synthesis was not required to mediate the retinoic acid repression of TIMP-1 mRNA levels and supports the evidence that retinoic acid acts at the transcriptional level to reduce TIMP-1 expression. In contrast, TGF-beta 1 increased TIMP-1 mRNA levels by 3.5-fold at 24 hr to > 10-fold at 72 hr without alterations in mRNA stability indicating that transforming growth factor (TGF)-beta 1 also acts at the transcriptional level to upregulate TIMP-1 expression in bone cells. Thus, these studies have revealed that TIMP-1 regulation by retinoic acid is different in osteoblasts from other cells and that retinoic acid has the property of generating resorptive and formative cell phenotypes in a tissue-specific manner. In bone, reduced TIMP-1 expression would favor bone matrix degradation and bone resorption that is a characteristic action of retinoids.
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PMID:Repression of tissue inhibitor of matrix metalloproteinase expression by all-trans-retinoic acid in rat bone cell populations: comparison with transforming growth factor-beta 1. 779 Mar 89

The mechanism by which Rev facilitates the export, and consequently, the translation of the structural protein mRNAs of the human immunodeficiency virus type 1 remains undefined. Previous immunolocalization has determined that Rev is predominantly in the nucleus with significant accumulation in the nucleolus, a localization consistent with the assumed site of Rev action. To determine whether the subcellular distribution is more dynamic than what was indicated by the original studies, the capacity of Rev to shuttle between the nucleus and cytoplasm was examined. It was observed that treatment of cells with DRB or actinomycin D resulted in a dramatic alteration in Rev distribution, the majority of the protein being found in the cytoplasm. Removal of the drug resulted in a rapid accumulation of Rev in the nucleus indicating that the block to nuclear import was reversible. Subsequent studies indicated that the movement of Rev into the cytoplasm was a passive process while its accumulation in the nucleus was an active one, given that only the latter displayed sensitivity to temperature. Finally, it was demonstrated that, while extensive redistribution of Rev could be attained by inhibition of RNA polymerase I alone, Rev was still capable of inducing expression of HIV structural gene expression under these conditions. Consequently, Rev activity does not appear to be dependent on either an intact nucleolus or the accumulation of the protein in the nucleus.
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PMID:HIV-1 Rev is capable of shuttling between the nucleus and cytoplasm. 809 47

The HIV-1 Tat protein enhances the formation of productive RNA polymerase II elongation complexes, potentially acting through a positive-acting, DRB-sensitive elongation factor. Tat is usually recruited to the HIV-1 promoter through the Tat trans-activation response element RNA stem-loop structure; however, recent data suggest that in certain cell types it can be directed instead through upstream enhancer elements. New studies also reveal that the response element overlaps a novel motif that promotes the assembly of abortive elongation complexes in the absence of Tat.
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PMID:Tat and the HIV-1 promoter. 835 64

P-TEFb is a key regulator of the process controlling the processivity of RNA polymerase II and possesses a kinase activity that can phosphorylate the carboxy-terminal domain of the largest subunit of RNA polymerase II. Here we report the cloning of the small subunit of Drosophila P-TEFb and the finding that it encodes a Cdc2-related protein kinase. Sequence comparison suggests that a protein with 72% identity, PITALRE, could be the human homolog of the Drosophila protein. Functional homology was suggested by transcriptional analysis of an RNA polymerase II promoter with HeLa nuclear extract depleted of PITALRE. Because the depleted extract lost the ability to produce long DRB-sensitive transcripts and this loss was reversed by the addition of purified Drosophila P-TEFb, we propose that PITALRE is a component of human P-TEFb. In addition, we found that PITALRE associated with the activation domain of HIV-1 Tat, indicating that P-TEFb is a Tat-associated kinase (TAK). An in vitro transcription assay demonstrates that the effect of Tat on transcription elongation requires P-TEFb and suggests that the enhancement of transcriptional processivity by Tat is attributable to enhanced function of P-TEFb on the HIV-1 LTR.
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PMID:Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro. 933 25

The transition from abortive into productive elongation is proposed to be controlled by a positive transcription elongation factor b (P-TEFb) through phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. Drosophila P-TEFb was identified recently as a cyclin-dependent kinase (CDK9) paired with a cyclin subunit (cyclin T). We demonstrate here the cloning of multiple cyclin subunits of human P-TEFb (T1 and T2). Cyclin T2 has two forms (T2a and T2b) because of alternative splicing. Both cyclin T1 and T2 are ubiquitously expressed. Immunoprecipitation and immunodepletion experiments carried out on HeLa nuclear extract (HNE) indicated that cyclin T1 and T2 were associated with CDK9 in a mutually exclusive manner and that almost all CDK9 was associated with either cyclin T1 or T2. Recombinant CDK9/cyclin T1, CDK9/cyclin T2a, and CDK9/cyclin T2b produced in Sf9 cells possessed DRB-sensitive kinase activity and functioned in transcription elongation in vitro. Either cyclin T1 or T2 was required to activate CDK9, and the truncation of the carboxyl terminus of the cyclin reduced, but did not eliminate, P-TEFb activity. Cotransfection experiments indicated that all three CDK9/cyclin combinations dramatically activated the CMV promoter.
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PMID:Identification of multiple cyclin subunits of human P-TEFb. 949 9


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