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)

Two promoter elements, the TATA element and initiator (Inr), are capable of directing specific transcription initiation of protein-encoding genes by RNA polymerase II (RNAPII). Although binding to the TATA element by the TATA-binding protein (TBP) has been shown to be the initial recognition step in transcription complex formation in vitro, the mechanism through which the basal machinery assembles into a functional complex on TATA-less promoters is controversial. Evidence supporting numerous models of Inr-mediated transcription complex formation exists, including the nucleation of a complex by Inr-binding proteins, a component of the TFIID complex, or a specific upstream activator common to many TATA-less promoters, Sp1. Using various techniques, we have undertaken a systematic analysis of the natural TATA-less human DNA polymerase beta (beta-pol) gene promoter. Although the beta-pol promoter contains upstream Sp1 elements and a functional Inr that binds YY1, neither of these factors is essential for Inr-mediated transcription complex formation. A complex containing TBP, TFIIB, TFIIF, and RNAPII (DBPolF complex) is capable of forming on the promoter in an Inr-dependent manner. A single point mutation within the Inr that affects DBPolF complex formation diminishes beta-pol transcriptional activity.
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PMID:Accurate positioning of RNA polymerase II on a natural TATA-less promoter is independent of TATA-binding-protein-associated factors and initiator-binding proteins. 915 95

The human gene for the seventh largest subunit of RNA polymerase II complex, hsRPB7 was cloned, sequenced and mapped. This complex is an integral part of the transcription-coupled DNA repair mechanism and has been shown to be involved in several human genetic diseases and implicated in many others. The hsRPB7 gene consists of 8 exons and spans approximately 5.1 kb. Southern blots of genomic and cloned DNA suggest that hsRPB7 is coded for by a single gene. Using human radiation hybrids and YACs, the gene was localized to 11q13.1, within 70 kb of marker D11S1765. The sequence of the 5' flanking region does not contain a TATA element, but does contain several Sp1 binding sites, an AP-1 site and a novel inverted polymorphic GATA tandem repeat. This novel GATA repeat can be used for linkage analysis. The hsRPB7 gene seems to be highly conserved among eukaryotic species, showing general sequence conservation to yeast and Drosophila. Northern blot analysis reveals a high degree of tissue-specific expression. For example, adult retina, brain and kidney exhibit a relatively high level of expression. A moderate level of expression is observed in heart, lung, testis, cornea, retinal pigmented epithelium/choroid and placenta with a lower level of expression in the uterus, small intestine and skeletal muscle. A very low level of expression was observed in stomach and liver. Comparison between four fetal and adult tissues also demonstrate a surprising level of developmental specificity. Expression in fetal retina is considerably lower than fetal brain but similar to adult retina.
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PMID:Human gene for the RNA polymerase II seventh subunit (hsRPB7): structure, expression and chromosomal localization. 925 63

DNA-dependent protein kinase (DNA-PK) has been known to catalyze phosphorylation of a number of regulatory factors involved in DNA replication and transcription such as simian virus 40 T antigen, p53, c-Myc, Sp1, and RNA polymerase II (Pol II). We examined the possibility that DNA-PK phosphorylates the general transcription factors TATA-binding protein (TBP) and transcription factor (TF) IIB, which play key roles in the formation of transcription initiation complex with Pol II. By using a highly purified preparation of DNA-PK from Raji cells, both TBP and TFIIB were shown to be phosphorylated in vitro by DNA-PK. We then investigated the effect of the phosphorylation of these factors on Pol II basal transcription. Stepwise analysis of preinitiation complex formation by electrophoretic mobility shift assay revealed that the phosphorylation of TBP and TFIIB by DNA-PK did not affect the formation of promoter (P)-TBP and P-TBP-TFIIB complexes but synergistically stimulated the formation of P-TBP-TFIIB-TFIIF-Pol II complex. Similarly, combination of the phosphorylated TBP and TFIIB synergistically stimulated Pol II basal transcription from adenovirus major late promoter. These observations suggest that DNA-PK could positively regulate the Pol II basal transcription by phosphorylating TBP and TFIIB.
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PMID:Phosphorylation of human general transcription factors TATA-binding protein and transcription factor IIB by DNA-dependent protein kinase--synergistic stimulation of RNA polymerase II basal transcription in vitro. 928 44

Affinity chromatography on columns containing the immobilized monomeric transcriptional elongation factor TFIIS or the essential large subunit, Elongin A, of the trimeric elongation factor, Elongin, was used to purify a human RNA polymerase II holoenzyme from HeLa whole cell extract. This holoenzyme contained nearstoichiometric amounts of all the general transcription factors, TFIIB, TFIID (TBP + TAFIIs), TFIIE, TFIIF, and TFIIH, required to accurately initiate transcription in vitro at the adenovirus major late promoter. It behaved as a large complex, slightly smaller than 70 S ribosomes, during gel filtration chromatography, and contained nearly half the TFIID that was present in the extract used for the affinity chromatography. It also contained the cyclin-dependent kinase CDK8, a human homologue of the Saccharomyces cerevisiae holoenzyme subunit SRB10, and many other polypeptides. Efficient interaction of holoenzyme with TFIIS or Elongin A required only the amino-terminal region of either protein. These regions are similar in amino acid sequence but dispensable for TFIIS or Elongin to regulate elongation in vitro by highly purified RNA polymerase II. The transcriptional activators GAL4-VP16 and GAL4-Sp1 activated transcription in vitro by purified holoenzyme in the absence of any additional factors.
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PMID:Interaction of elongation factors TFIIS and elongin A with a human RNA polymerase II holoenzyme capable of promoter-specific initiation and responsive to transcriptional activators. 930 22

Representatives of three distinct classes of mammalian protein domain activating RNA polymerase II were fused to the yeast GAL4p DNA-binding domain. The resulting fusion proteins were tested in the fission yeast Schizosaccharomyces pombe for their ability to activate transcription of different reporter constructs containing GAL4-binding sites in positions close to or far from the TATA box. The acidic-rich activation domain of VP16 stimulates transcription in S.pombe from proximal and distal positions, suggesting that the mechanism of activation is conserved from man to budding and fission yeasts. Unlike in Saccharomyces cerevisiae, the glutamine-rich activation domains of Sp1, Oct1 and Oct2 activate transcription in S. pombe when tested in a proximal TATA box context. Similarly to mammalian cells, these domains are inactive or weakly active when tested in a distal position. Moreover, the proline-rich activation domains of AP-2 and CTF/NF1 display strong transcriptional activities from a TATA box-proximal position, and weak activities when tested in a remote position. Consequently, proline-rich and glutamine-rich activation domains act differently in S.cerevisiae and mammalian cells, but similarly in S.pombe and mammalian cells.
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PMID:Three classes of mammalian transcription activation domain stimulate transcription in Schizosaccharomyces pombe. 931 30

Multiple transcriptional start sites have been identified for the gene encoding the rat Type I isozyme of hexokinase (White, J.A., Liu, W., and Wilson, J. E., Arch. Biochem. Biophys. 335, 161-172, 1996); these are clustered at positions approximately -460, -300, and -100 relative to the translational start codon (ATG, with A being +1). PC12 cells and H9c2 cells were transfected with luciferase reporter constructs containing genomic sequence between positions -3366 and -171. Marked (85%) decrease in promoter activity was associated with deletion of sequence between -742 and -516. In DNase I footprinting experiments, two regions, called P1 (-552 to -529) and P2 (-480 to -458) boxes, were protected by proteins present in nuclear extracts from PC12 cells. Mutation or deletion of the P2 box had no effect on promoter activity; protection in this region, which includes the most upstream cluster of transcriptional start sites, is attributed to binding of RNA polymerase II or associated factors. In contrast, mutations or deletions in the P1 box had markedly detrimental effects on promoter activity and on binding of proteins in PC12 cell nuclear extracts. Maintenance of a consensus Sp1 binding site centrally located in the P1 box was critical for both promoter activity and binding. A second Sp1 site (-570), just upstream from the P1 box, was also shown to be functionally important but no protection of this region was detected in footprinting experiments, presumably reflecting lower affinity at this site under the conditions used. Supershift experiments demonstrated the involvement of Sp1, Sp3, and Sp4 in formation of complexes with the P1 box region and implicate these transcription factors in regulating promoter activity associated with this region. Another series of reporter constructs, including sequence between -171 and -1, permitted detection of an additional promoter activity downstream from -364. While not yet extensively characterized, it is already evident that the cis elements influencing the downstream promoter activity are distinct from the Sp factors determined to be important in expression from the upstream promoter region.
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PMID:Two Sp sites are important cis elements regulating the upstream promoter region of the gene for rat type I hexokinase. 932 94

Many eukaryotic RNA polymerase II promoters contain initiator elements which direct accurate transcription in a TATA-independent manner. The PEA3/Ets-binding site (PEA3/EBS) is a common enhancer element in eukaryotic genes and is also found near the transcriptional start sites of many TATA-less promoters. We demonstrate that two PEA3/EBSs driving expression of the luciferase reporter gene, function as a minimal transcriptional initiator element. Maximal levels of transcription was achieved when two PEA3/EBSs, in either orientation, were located on the same face of the DNA helix, and the sites could be separated by up to three helical turns. In vitro transcription start sites directed by PEA3/EBS elements were clustered on either side of the upstream PEA3/EBS and were abolished by immunodepletion of GA-binding protein (GABP) from FM3A cell nuclear extracts. In vivo, co-transfection of GABPalpha and GABPbeta expression vectors enhanced reporter gene expression driven from PEA3/EBS initiator elements. Like other initiator elements, the PEA3/EBS elements were activated synergistically by upstream Sp1-binding sites. Thus, our results establish GABP as both a transcriptional activator factor and as an initiator factor.
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PMID:GA-binding protein-dependent transcription initiator elements. Effect of helical spacing between polyomavirus enhancer a factor 3(PEA3)/Ets-binding sites on initiator activity. 936 Sep 80

Tat of HIV-2 (Tat-2) requires host cellular factors for optimal function. We show that transactivation by Tat-2 of the HIV promoter requires cis-acting binding sites for Sp1 or Sp1 brought to the promoter via a heterologous system. We demonstrate that an activation domain in Tat-2 consists of one of two potential alpha-helices in the amino-terminal region, the cysteine-rich region, and the core region and that this independent activation domain requires cis-acting Sp1-binding sites for function. Tat-2 interacts with Sp1 in in vitro binding assays, and these interactions require basic residues outside of the Tat-2 activation domain. The regions in Sp1 sufficient for functional synergy with Tat are the Sp1 activation domains, while the DNA-binding region is dispensable. Substitution mutations of a glutamine-rich region in one Sp1 activation domain, which eliminate interactions with a TBP-associated factor, also significantly decrease synergy with Tat. Thus, the functional synergy between Tat-2 and Sp1 localizes to domains in each activator that interact with components of the transcription complex. We suggest that these interactions, rather than direct Tat/Sp1 binding, result in highly processive RNA polymerase II complexes and full-length viral transcripts.
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PMID:Interactions between Tat of HIV-2 and transcription factor Sp1. 940 May 95

Ankyrin 1, an erythrocyte membrane protein that links the underlying cytoskeleton to the plasma membrane, is also expressed in brain and muscle. We cloned a truncated, muscle-specific ankyrin 1 cDNA composed of novel 5' sequences and 3' sequences previously identified in the last 3 exons of the human ankyrin 1 erythroid gene. Northern blot analysis revealed expression restricted to cardiac and skeletal muscle tissues. Deduced amino acid sequence of this muscle cDNA predicted a peptide of 155 amino acids in length with a hydrophobic NH2 terminus. Cloning of the corresponding chromosomal gene revealed that the ankyrin 1 muscle transcript is composed of four exons spread over approximately 10 kilobase pairs of DNA. Reverse transcriptase-polymerase chain reaction of skeletal muscle cDNA identified multiple cDNA isoforms created by alternative splicing. The ankyrin 1 muscle promoter was identified as a (G + C)-rich promoter located > 200 kilobase pairs from the ankyrin 1 erythroid promoter. An ankyrin 1 muscle promoter fragment directed high level expression of a reporter gene in cultured C2C12 muscle cells, but not in HeLa or K562 (erythroid) cells. DNA-protein interactions were identified in vitro at a single Sp1 and two E box consensus binding sites contained within the promoter. A MyoD cDNA expression plasmid transactivated an ankyrin 1 muscle promoter fragment/reporter gene plasmid in a dose-dependent fashion in both HeLa and K562 cells. A polyclonal antibody raised to human ankyrin 1 muscle-specific sequences reacted with peptides of 28 and 30 kDa on immunoblots of human skeletal muscle.
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PMID:An alternate promoter directs expression of a truncated, muscle-specific isoform of the human ankyrin 1 gene. 943 Jun 67

Transcriptional interference between adjacent genes has been demonstrated in a variety of biological systems. To study this process in RNA polymerase II (pol II) transcribed genes we have analysed the effect of transcription on tandem HIV-1 promoters integrated into the genome of HeLa cells. We show that transcriptional activation at the upstream promoter reduces transcription from the downstream promoter, as compared with basal transcription conditions (in the absence of an activator). Furthermore, insertion of a strong transcriptional termination element between the two promoters alleviates this transcriptional interference, resulting in elevated levels of transcription from the downstream promoter. Actual protein interactions with the downstream (occluded) promoter were analysed by in vivo footprinting. Binding of Sp1 transcription factors to the occluded promoter was reduced, when compared with the footprint pattern of the promoter protected by the terminator. This suggests that promoter occlusion is due to disruption of certain transcription factors and that it can be blocked by an intervening transcriptional terminator. Chromatin mapping with DNase I indicates that a factor binds to the termination element under both basal and induced conditions.
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PMID:Transcriptional interference perturbs the binding of Sp1 to the HIV-1 promoter. 946 40


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