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 functions of most of the 12 subunits of the RNA polymerase II (Pol II) enzyme are unknown. In this study, we demonstrate that two of the subunits, hRPB2 and hRPB10alpha, mediate the regulated stimulation of transcription. We find that the transcriptional coactivator BRCA1 interacts directly with the core Pol II complex in vitro. We tested whether single subunits from Pol II would compete with the intact Pol II complex to inhibit transcription stimulated by BRCA1. Excess purified Pol II subunits hRPB2 or hRPB10alpha blocked BRCA1- and VP16-dependent transcriptional activation in vitro with minimal effect on basal transcription. No other Pol II subunits tested inhibited activated transcription in these assays. Furthermore, hRPB10alpha, but not hRPB2, blocked Sp1-dependent activation.
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PMID:BRCA1 interaction with RNA polymerase II reveals a role for hRPB2 and hRPB10alpha in activated transcription. 1072 6

In yeast cells, transcriptional activation occurs when the RNA polymerase II (Pol II) machinery is artificially recruited to a promoter by fusing individual components of this machinery to a DNA-binding domain. Here, we show that artificial recruitment of components of the TFIID complex can activate transcription in mammalian cells. Surprisingly, artificial recruitment of TATA-binding protein (TBP) activates transiently transfected and chromosomally integrated promoters with equal efficiency, whereas artificial recruitment of TBP-associated factors activates only chromosomal reporters. In contrast, artificial recruitment of various components of the mammalian Pol II holoenzyme does not confer transcriptional activation, nor does it result in synergistic activation in combination with natural activation domains. In the one case examined in more detail, the Srb7 fusion failed to activate despite being associated with the Pol II holoenzyme and being directly recruited to the promoter. Interestingly, some acidic activation domains are less effective when the promoter is chromosomally integrated rather than transiently transfected, whereas the Sp1 glutamine-rich activation domain is more effective on integrated reporters. Thus, yeast and mammalian cells differ with respect to transcriptional activation by artificial recruitment of the Pol II holoenzyme.
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PMID:Artificial recruitment of TFIID, but not RNA polymerase II holoenzyme, activates transcription in mammalian cells. 1082 98

The TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s) compose the general transcription factor TFIID. The TAF(II) subunits mediate activated transcription by RNA polymerase II by interacting directly with site-specific transcriptional regulators. TAF(II)s also participate in promoter recognition by contacting core promoter elements in the context of TFIID. To further dissect the contribution of individual TAF(II) subunits to mammalian TFIID function, we employed a vaccinia virus-based protein expression system to study protein-protein interactions and complex assembly. We identified the domains of human (h) TAF(II)130 required for TAF(II)-TAF(II) interactions and formation of a complex with hTBP, hTAF(II)100, and hTAF(II)250. Functional analysis of partial TFIID complexes formed in vivo indicated that hTAF(II)130 was required for transcriptional activation by Sp1 in vitro. DNase I footprinting experiments demonstrated that purified hTBP/hTAF(II)250 complex reconstituted with or without additional TAF(II)s was significantly reduced for TATA box binding (as much as 9-fold) compared with free hTBP. By contrast, hTAF(II)130 stabilized binding of hTBP to the TATA box, whereas hTAF(II)100 had little effect. Thus, our biochemical analysis supports the notion that TAF(II)s possess distinct functions to regulate the activity of TFIID.
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PMID:Assembly of partial TFIID complexes in mammalian cells reveals distinct activities associated with individual TATA box-binding protein-associated factors. 1089 37

To investigate the role of chromatin structure in the regulation of transcription by RNA polymerase II, we developed a chromatin transcription system in which periodic nucleosome arrays are assembled with purified recombinant ATP-utilizing chromatin assembly and remodeling factor (ACF), purified recombinant nucleosome assembly protein 1 (dNAP1), purified native core histones, plasmid DNA, and ATP. With this chromatin, we observed robust activation of transcription with three different transcription factor sets (nuclear factor kappaB p65 + Sp1, estrogen receptor, and Gal4-VP16) added either before or after chromatin assembly. In fact, the efficiency of activated transcription from the ACF + dNAP1-assembled chromatin was observed to be comparable with that from naked DNA templates or chromatin assembled with a crude Drosophila extract (S190). With ACF + dNAP1-assembled chromatin, we found that transcriptional activation is dependent upon acetyl-CoA. This effect was not seen with naked DNA templates or with crude S190-assembled chromatin. We further determined that acetyl-CoA is required at the time of preinitiation complex assembly but not during assembly of the chromatin template. These findings suggest that there is at least one key acetylation event that is needed to assemble a functional transcription preinitiation complex with a chromatin template.
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PMID:Transcriptional analysis of chromatin assembled with purified ACF and dNAP1 reveals that acetyl-CoA is required for preinitiation complex assembly. 1105 7

The proficiency with which anthracyclines and other DNA-binding drugs target certain sequences in eukaryotic promoters offers a potential approach to interfere with the mechanisms that regulate gene expression in tumor cells. An in vitro transcription assay has been used to compare the ability of the bisintercalating anthracycline WP631 and the monointercalating anthracycline daunorubicin in terms of their ability to inhibit initiation of transcription of the adenovirus major late promoter linked to a G-less transcribed DNA template. Both drugs inhibit basal transcription by RNA polymerase II. However, WP631 is approximately 15 times more efficient at inhibiting transcription initiation from an adenovirus promoter containing an upstream Sp1-protein binding site. The differences in the ability of each drug to inhibit transcription initiation appear to be related to the competition between Sp1 and the anthracyclines for binding to the same site. To see whether WP631's strong effect on transcription can also be observed in cells, we compared the effects of WP631 and other anthracyclines on the transcription of the c-myc gene, which promoter contains Sp1 binding sites. The resulting data suggest that WP631 might circumvent some kinds of tumor resistance at rather low drug concentrations, inhibit c-myc expression in some cell lines, and exert its antitumoral effect by inducing apoptosis.
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PMID:Analysis of the effects of daunorubicin and WP631 on transcription. 1117 87

Telomerase, the reverse transcriptase that maintains telomere DNA, is usually undetectable in adult human tissues, but is positive in embryonic tissues and in cancers. However, in rodents, several organs of normal adult animals express substantial amounts of telomerase activity. To elucidate relevant control mechanisms operating on the tissue-specific expression of telomerase in rodents, we examined the transcriptional regulation of telomerase reverse transcriptase (mTERT) gene in muscle cell differentiation. Reverse transcriptase-polymerase chain reaction analysis showed that the reduction of telomerase activity was caused by the decrease of mTERT mRNA level during myogenesis. Transfections of mTERT promoter showed that the proximal 225-base pair region is the core promoter responsible for basal transcriptional activity and also participates in the reduced transcription after muscle differentiation. Electrophoretic mobility shift assays showed that this region contained the GC-boxes, which bind to Sp1 family proteins, and the E-box, which binds to c-Myc. Furthermore, DNA binding activities of Sp1, Sp3, and c-Myc were down-regulated during myogenesis. These data suggest that Sp1, Sp3, and c-Myc have critical roles of TERT transactivation in mouse, and the lack of these transcription factors cause down-regulation of mTERT gene expression in muscle cells differentiation.
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PMID:Mechanism for the reduction of telomerase expression during muscle cell differentiation. 1127 34

Indole-3-carbinol (I3C), a compound naturally occurring in Brassica vegetables, can induce a G(1) cell cycle arrest of human MCF-7 breast cancer cells that is accompanied by the selective inhibition of cyclin-dependent kinase 6 (CDK6) expression. Reverse transcriptase-polymerase chain reaction analysis of CDK6 mRNA decay rates revealed that I3C had no effect on CDK6 transcript stability. We report the first identification and functional characterization of the CDK6 promoter in order to determine whether I3C inhibits CDK6 transcription. In MCF-7 cells stably transfected with CDK6 promoter-linked luciferase reporter plasmids, I3C inhibited CDK6 promoter activity in an I3C-specific response that was not a consequence of the growth-arrested state of the cells. Deletion analysis revealed a 167-base pair I3C-responsive region of the CDK6 promoter between -805 and -638. Site-specific mutations within this region revealed that both Sp1 and Ets-like sites, which are spaced 5 base pairs apart, were necessary for I3C responsiveness in the context of the CDK6 promoter. Electrophoretic mobility shift analysis of protein-DNA complexes formed with nuclear proteins isolated from I3C-treated and -untreated cells, in combination with supershift assays using Sp1 antibodies, demonstrated that the Sp1-binding site in the CDK6 promoter forms a specific I3C-responsive DNA-protein complex that contains the Sp1 transcription factor. Taken together, our results suggest that I3C down-regulates CDK6 transcription by targeting Sp1 at a composite DNA site in the CDK6 promoter.
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PMID:Indole-3-carbinol inhibits CDK6 expression in human MCF-7 breast cancer cells by disrupting Sp1 transcription factor interactions with a composite element in the CDK6 gene promoter. 1129 39

The posttranslational modification of eukaryotic intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc) monosaccharides is essential for cell viability, yet its precise functional roles are largely unknown. O-GlcNAc transferase utilizes UDP-GlcNAc, the end product of hexosamine biosynthesis, to catalyze this modification. The availability of UDP-GlcNAc correlates with glycosylation levels of intracellular proteins as well as with transcriptional levels of some genes. Meanwhile, transcription factors and RNA polymerase II can be modified by O-GlcNAc. A linkage between transcription factor O-GlcNAcylation and transcriptional regulation therefore has been postulated. Here, we show that O-GlcNAcylation of a chimeric transcriptional activator containing the second activation domain of Sp1 decreases its transcriptional activity both in an in vitro transcription system and in living cells, which is in concert with our observation that O-GlcNAcylation of Sp1 activation domain blocks its in vitro and in vivo interactions with other Sp1 molecules and TATA-binding protein-associated factor II 110. Furthermore, overexpression of O-GlcNAc transferase specifically inhibits transcriptional activation by native Sp1 in cells. Thus, our studies provide direct evidence that O-GlcNAcylation of transcription factors is involved in transcriptional regulation.
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PMID:O-linkage of N-acetylglucosamine to Sp1 activation domain inhibits its transcriptional capability. 1137 15

The effects of DNA interacting drugs on: (1) total RNA synthesis catalyzed by E. coli and T7 RNA polymerase; (2) synthesis of the initiating dinucleotide (pppApU) by E. coli RNA polymerase ("abortive initiation"); (3) elongation of RNA chains synthesized by T7 RNA polymerase on pT7-7 plasmid DNA bearing T7 RNA polymerase promoter phi 10 with human Cu/Zn superoxide dismutase coding sequence, (4) interaction of transcription factor Sp1 and its binding site were studied. Intercalating ligands which form quickly dissociating complexes with DNA (anthracyclines, proflavine, ethidium bromide) are compared with the slowly dissociating drug of d(G x C) specificity (actinomycin D), the non-intercalating, d(A x T) specific pyrrole antibiotics (netropsin and distamycin A) and covalently binding to DNA 1-nitroacridine derivative (nitracrine). The obtained results indicate that rapidly dissociating ligands, proflavine and ethidium bromide, inhibit total RNA synthesis in vitro and the abortive initiation to a similar extent while they do not induce discrete elongation stops of RNA polymerase. Actinomycin D and nitracrine exhibit a high inhibitory effect on total RNA synthesis and induce stops of RNA polymerase while not affecting abortive initiation. Pyrrole antibiotics primarily inhibit the initiation, while no elongation stops are induced. Actinomycin D inhibits complex formation between nuclear proteins and the Sp1 binding site. Netropsin, ethidium bromide, proflavine and other intercalating acridines do not affect Sp1 binding. The results indicate that the effects primarily depend on sequence specificity and secondarily on the dissociation rate of ligands from their complexes with DNA.
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PMID:Effects of anticancer drugs on transcription in vitro. 1172

The general transcription factor TFIID facilitates recruitment of the transcription machinery to gene promoters and regulates initiation of transcription by RNA polymerase II. hTAF(II)130, a component of TFIID, interacts with and serves as a coactivator for multiple transcriptional regulatory proteins, including Sp1 and CREB. A yeast two-hybrid screen has identified an interaction between hTAF(II)130 and heterochromatin protein 1 (HP1), a chromatin-associated protein whose function has been implicated in gene silencing. We find that hTAF(II)130 associates with HP1 in an isoform-specific manner: HP1alpha and HP1gamma bind to hTAF(II)130, but not HP1beta. In addition, we show that endogenous hTAF(II)130 and components of TFIID in HeLa nuclear extracts associate with glutathione S-transferase-HP1alpha and -HP1gamma. hTAF(II)130 possesses a pentapeptide HP1-binding motif, and mutation of the hTAF(II)130 HP1 box compromises the interaction of hTAF(II)130 with HP1. We demonstrate that Gal4-HP1 proteins interfere with hTAF(II)130-mediated activation of transcription. Our results suggest that HP1alpha and HP1gamma associate with hTAF(II)130 to mediate repression of transcription, supporting a new model of transcriptional repression involving a specific interaction between a component of TFIID and chromatin.
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PMID:Isoform-specific interaction of HP1 with human TAFII130. 1195 14


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