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)

We previously identified murine DNA sequences that stimulate the amplification of cis-linked plasmid DNA in mouse cells under selective conditions (Holst et al., 1988). Here we focus on the structural features of one of these elements, the 229-bp element 5. The amplification-promoting activity was fully recovered from the middle part of element 5. The active region interacted in a sequence-specific way with a protein from nuclear extracts. Using footprinting analyses the binding region was characterized and subsequently shown to be functionally active as an amplification-promoting sequence, whereas a mutated binding site was inactive. Therefore, cis-acting element 5 functioned via interaction with a trans-acting factor. The same binding site was also active as a promoter element for RNA polymerase II transcription, because it efficiently reconstituted the activity of a truncated herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene promoter lacking the distal Sp1 binding site. Thus, the same protein seems to function in both RNA polymerase II transcription and DNA amplification. Possible relationships between both functions are discussed.
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PMID:An amplification-promoting sequence from mouse genomic DNA: interaction with a trans-acting factor that also affects gene expression. 237 75

The distribution of binding sites for the ultimate carcinogen anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE-l) in the 5' region of the Chinese hamster ovary aprt gene has been determined. A plasmid (pGAL) containing the entire hamster aprt gene including the 3' and 5' flanking regions was inserted into the BamHI site of the multiple cloning site of pGEM so that the T7 promoter was 5' to the aprt gene. In vitro transcription of BPDE-I-modified pGAL, using the T7 RNA polymerase, revealed two prominent transcriptional stop sites. One of these sites was located in the first exon of the aprt gene, whereas the second transcriptional stop was located approximately 150 bp upstream from the translational start site. This latter region contains two perfect GC-box consensus sequences that are potential Sp1 binding sites. Using a specific laser cutting technique to map BPDE-I DNA binding sites in the 5' flanking region of the aprt gene, we found that the DNA region containing the GC-box consensus sequences was indeed a hot spot for BPDE-I modification.
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PMID:Preferential modification of GC boxes by benzo[a]pyrene-7,8-diol-9,10-epoxide. 250 85

The Epstein-Barr virus-encoded small RNA (EBER) genes appear to comprise an interesting subset of class III genes different from any previously identified, including U6 and 7SK. EBER genes have functional A and B box intragenic control regions. In addition, they contain three upstream elements that together stimulate in vivo expression 50-fold and resemble sites associated with typical class II promoters. DNAase I footprinting analyses using purified proteins or oligonucleotide competition demonstrate that nucleotides -40 to -55 bind activating transcription factor (ATF) or a related protein, while nucleotides -56 to -77 bind Sp1 protein or a related protein. The element between positions -23 and -28 resembles a TATA box. EBERs are unusual RNA polymerase III transcripts shown to be controlled by ATF- and Sp1-like promoter elements, suggesting mechanisms for their high level expression in EBV-transformed lymphocytes.
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PMID:Epstein-Barr virus small RNA (EBER) genes: unique transcription units that combine RNA polymerase II and III promoter elements. 254 26

We recently found that many RNA polymerase II transcription factors are modified with N-acetylglucosamine residues. These sugar moieties confer upon transcription factors an ability to bind the lectin wheat germ agglutinin. We have taken advantage of this interaction to devise a purification procedure for the "GC-box" binding transcription factor Sp1. Crude nuclear extracts are first subjected to wheat germ agglutinin affinity chromatography and then subjected to sequence-specific DNA affinity chromatography. The Sp1 protein purified by this procedure is at least 95% pure, and the overall recovery is greater than 80%. In addition to yielding larger quantities of Sp1 than conventional schemes, the new purification procedure is also simpler and more rapid. We show that wheat germ agglutinin affinity chromatography can also be used to purify the glycosylated forms of the CCAAT-binding transcription factor. Thus, wheat germ agglutinin affinity chromatography may aid the purification of other transcription factors that bear N-acetylglucosamine residues. Furthermore, the ability to separate glycosylated forms of transcription factors from their unglycosylated counterparts by wheat germ agglutinin affinity chromatography should facilitate investigations into the role of N-acetylglucosamine residues in the functioning of transcription factor proteins.
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PMID:Purification and analysis of RNA polymerase II transcription factors by using wheat germ agglutinin affinity chromatography. 264 81

We have studied the interactions of the Sp1 and IID transcription factors with a simple RNA polymerase II promoter. The adenovirus E1B core promoter consists essentially of a GC box and a TATA box, binding sites for the Sp1 and IID transcription factors, respectively. The E1B promoter is accurately transcribed in vitro using a mammalian transcription system. Sp1 activates E1B transcription in vitro in reactions using IID factor isolated from either human or yeast cells. In DNase I footprinting studies, Sp1 bound rapidly to its recognition sequence even at 0 degrees C (t1/2 less than 1 min). In contrast, yeast IID bound more slowly (t1/2 approximately 6 min at 25 degrees C) and required thermal energy for stable binding to the TATA box sequence. Dissociation rates were measured by the addition of specific oligonucleotide competitors to preformed DNA-protein complexes. Sp1 dissociates rapidly (t1/2 less than 1 min) at 25 degrees C, while yeast IID dissociates with an estimated t1/2 of 1 h at 25 degrees C. Sp1 and yeast IID bound to the E1B promoter simultaneously but independently. The rates of binding and dissociation of these factors were not significantly affected by the presence of the other factor. Bound Sp1 factor did not alter or enhance the yeast IID footprint. Oligonucleotide challenge of in vitro transcription reactions indicated that Sp1 also did not enhance the binding of the human IID factor to the E1B promoter. Thus the Sp1 factor activates transcription of the E1B gene by a mechanism that does not enhance the DNA-binding activity of the IID factor. Sp1 factor activates E1B transcription by 5- to 10-fold in vitro. Under these in vitro transcription conditions, transcripts due to reinitiation from an individual promoter complex contribute only a small portion of the total yield of E1B transcripts. Thus Sp1 cannot activate transcription by increasing the rate of initiation events per complex. Instead it appears that Sp1 acts by increasing the number of productive transcription complexes formed in vitro.
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PMID:Sp1 activates transcription without enhancing DNA-binding activity of the TATA box factor. 267 69

The genomic locus (RPII215) encoding the largest subunit of mouse RNA polymerase II has been cloned by low stringency hybridization to a Drosophila RPII215 probe. The mouse gene consists of 28 exons which span 30 kilobases. Analysis of the nucleotide and predicted protein sequences indicates that the protein is comprised of two domains. There is a 1500 residue amino-terminal domain which contains seven regions strikingly similar to those in the beta' subunit of Escherichia coli RNA polymerase, and a carboxyl-terminal domain comprised of 52 repeats of a 7-amino-acid consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Among the seven highly conserved regions are a strongly basic domain consistent with a DNA-binding site and a consensus sequence characteristic of a potential zinc-binding domain. The 5' upstream region contains three tandem sequences similar to binding sites for the transcription factor SP1. Two of the introns in this gene splice at donor GC dinucleotides as opposed to previously described invariant GT sites. The identification of regions which are highly conserved as compared with bacterial and yeast RNA polymerase and other regions which are unique to the mouse protein suggests which domains of RNA polymerase large subunits are involved in aspects of transcription common to both procaryotes and eucaryotes and which are characteristic of transcription in higher organisms.
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PMID:Cloning and sequence analysis of the mouse genomic locus encoding the largest subunit of RNA polymerase II. 303 94

Sp1 is a sequence-specific DNA binding protein that activates RNA polymerase II transcription from promoters that contain properly positioned GC boxes. A series of deletion mutants of Sp1 were expressed in Escherichia coli and used to identify separate regions of the protein that are important for three different biochemical activities. The sequence-specificity of DNA binding was conferred by Zn(II) fingers, whereas a different region of Sp1 appeared to regulate the affinity of DNA binding. The E. coli-synthesized Sp1 was able to stimulate initiation of RNA synthesis in vitro, and at least two distinct segments of the protein contributed to its transcriptional activity.
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PMID:Distinct regions of Sp1 modulate DNA binding and transcriptional activation. 305 95

Glycosylation is often regarded as being restricted to proteins confined to the cell surface or within the lumen of intracellular organelles. Here we show that the human RNA polymerase II transcription factor Sp1 bears multiple O-linked N-acetylglucosamine (GlcNAc) monosaccharide residues. The lectin wheat germ agglutinin specifically inhibits the transcriptional activation but not the DNA binding function of Sp1. Furthermore, many other RNA polymerase II transcription factors also bear terminal GlcNAc residues, whereas most nuclear proteins, including RNA polymerase I and III transcription factors tested, do not. In some cases, only a subset of the polypeptide species within a particular family of closely related RNA polymerase II factors appears to be glycosylated. Our findings raise the possibility that O-linked GlcNAc residues play a role in the mechanism or regulation of transcriptional activation of RNA polymerase II.
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PMID:O-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation. 313 1

DNA polymerase beta (beta-pol) is a housekeeping enzyme considered to be involved in DNA repair in vertebrate cells. We cloned a fragment of genomic DNA spanning the first two exons of the human beta-pol gene and approximately 11 kilobases of the flanking region. The segment just 5' of the transcription start site can direct expression of the bacterial chloramphenicol acetyltransferase (CAT) gene in HeLa cells. A sequence containing only 113 base pairs of flanking DNA has promoter activity, and various constructs containing up to 4.8 kilobases of flanking sequence are expressed at a similar level, indicating that with this assay the important regulatory elements are located within or proximal to the approximately 100-bp core promoter. S1 nuclease mapping was used to show that transcription of the transfected genes is initiated at the same position as the endogenous beta-pol gene. The region upstream of the transcription start site is G + C rich and contains neither CAAT nor TATA boxes, but does have three decanucleotide elements matching high affinity binding sites for the RNA polymerase II transcription factor Sp1. Extending 5' from position -39 and surrounded by Sp1 consensus binding elements, there is a 10-nucleotide sequence with perfect dyad symmetry, GTGACGTCAC. Similar sequences are found in a number of cellular and viral promoters, including several adenovirus promoters. Experiments to test whether the core beta-pol promoter is activated by the adenovirus early region products showed that cotransfection with an adenovirus expression plasmid strongly activates expression of the beta-pol promoter.
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PMID:Human beta-polymerase gene. Structure of the 5'-flanking region and active promoter. 318 28

The human transcription factor Sp1 binds upstream of certain viral and cellular promoters and activates initiation of RNA synthesis from these promoters by RNA polymerase II. The Sp1 binding sites of both simian virus 40 and a related monkey promoter contain multiple copies of the sequence GGGCGG, three or four of these sequences forming close contacts with Spl. The clustered contacts fall on one strand of the DNA and are arranged similarly in the major groove of the DNA helix in both promoters.
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PMID:Multiple specific contacts between a mammalian transcription factor and its cognate promoters. 609


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