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 have analyzed the interaction of the general RNA polymerase II transcription factor TFIID with its DNA-binding site, the TATA box (consensus sequence TATAAAA). We have demonstrated that TFIID, unlike most sequence-specific DNA-binding proteins, interacts primarily within the minor groove of the DNA helix. This was established by a novel approach involving complete replacement of the thymines and adenines in the TATA box with cytosines and inosines, respectively. This substitution exchanged the major groove of TATAAAA for that of the sequence CGCGGGG, without altering the surface of the minor groove. The unusual DNA-binding properties of TFIID revealed by this study have important implications for TFIID specificity and function and, more generally, for sequence-specific recognition by DNA-binding proteins.
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PMID:TFIID binds in the minor groove of the TATA box. 176 Aug 47

Transcription of small genes by RNA polymerase III or C (pol III) involves many of the strategies that are used for transcription complex formation and occasionally the same components as those used by RNA polymerase II or B (pol II). Transcription complex formation is a multistep process that leads to the binding of a single initiation factor, TFIIIB, which in turn directs the selection of pol III. The general transcription factor TFIID can be involved in both pol II and pol III transcription. These and other similarities point towards a unifying mechanism for eukaryotic transcription initiation.
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PMID:RNA polymerase III (C) and its transcription factors. 177 70

We have identified a component of the eukaryotic RNA polymerase II transcriptional machinery that is more heat-labile than TFIID. DHFR transcriptional activity was severely reduced in 40 degrees C heat-treated extracts in which TFIID was fully active. This heat-labile activity was required for the transcription of both TATA box and non-TATA box promoters that are activated by the transcription factor Sp1. Gel mobility shifts indicated that Sp1 DNA binding activity was heat-labile, and the addition of purified Sp1 to 40 degrees C heat-treated extracts fully restored DHFR transcriptional activity. In contrast, the addition of Sp1 to 47 degrees C heat-treated extract did not result in transcriptional activity from the DHFR promoter. We conclude that reduction in Sp1 DNA binding activity is partially responsible for the heat-sensitive loss of DHFR transcriptional activity, but that a second essential activity is also inactivated by 47 degrees C heat-treatment. The discovery of this heat-labile component of Sp1 activation has two important implications in the analysis of transcriptional regulation. First, it demonstrates that heat-treated extracts are not appropriate for examination of the involvement of TFIID in the transcription of Sp1-activated promoters. Second, it explains the previously reported low-temperature optima for transcription from the DHFR promoter and demonstrates that transcriptional studies of Sp1-activated promoters should not be performed at 30 degrees C.
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PMID:Sp1 activation of RNA polymerase II transcription complexes involves a heat-labile DNA-binding component. 182 Feb 11

A T7 RNA polymerase expression system has been used for the efficient expression of the yeast RNA polymerase general transcription factor TFIID (TFIIDY), the TATA-box factor (previously called BTF1) in Escherichia coli. Expression of the gene was performed at 25 degrees C instead of 37 degrees C to increase the total amount of soluble TFIIDY. Soluble TFIIDY was purified in three chromatographic steps and was eluted from the final column, a heparin-5PW HPLC column, in two peaks at 0.38 M (peak I) and 0.42 M (peak II) KCl in which this protein was 52% and greater than 95% pure, respectively. The protein in both peaks was active in an in vitro transcription assay. However, while TFIIDY from peak II was essentially indistinguishable from the material isolated from yeast, the protein of peak I differed in a number of biochemical characteristics, having a lower specific activity in an in vitro transcription assay and displaying an altered pattern of bands in a DNA band shift assay. Despite these differences, the proteins in both peaks have identical molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, have indistinguishable N-terminal amino acid sequences, and apparently exist as monomers under the conditions used for the heparin-5PW chromatography.
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PMID:Expression in Escherichia coli: purification and properties of the yeast general transcription factor TFIID. 182 18

The herpes simplex virus type 1 (HSV-1) ICP4 protein is a transcriptional activator of many eucaryotic RNA polymerase II promoters. The HSV-1 thymidine kinase gene (tk) promoter is induced by ICP4 and contains binding sites for the cellular transcription factors TFIID, Sp1, and CCAAT-binding proteins, each of which affects expression of the tk gene. In this study, the effects of mutations in these sites on the transcription of tk in the presence and absence of ICP4 were determined during viral infection. Only the TATA box was necessary for efficient expression in the presence of ICP4; however, ICP4 apparently can still induce tk transcription even when the TATA box is disrupted. Alteration of the Sp1 sites had a minor effect on ICP4-induced expression in comparison to a large effect in the absence of ICP4, indicating that ICP4 can operationally substitute for the function of the transcription factor Sp1. In addition, tk was still expressed with the kinetics of an early gene in the absence of binding sites for Sp1 and CCAAT-binding proteins.
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PMID:Herpes simplex virus transactivator ICP4 operationally substitutes for the cellular transcription factor Sp1 for efficient expression of the viral thymidine kinase gene. 184 84

Mobility of P transposable elements in Drosophila melanogaster depends on the 87-kDa transposase protein encoded by the P element. Transposase recognizes a 10-base-pair DNA sequence that overlaps an A + T-rich region essential for transcription from the P-element promoter. We report here that transposase represses transcription from the P-element promoter in vitro. This transcriptional repression is blocked by prior formation of an RNA polymerase II transcription complex on the template DNA. Binding of transposase on the P-element promoter is blocked by prior binding of either the Drosophila RNA polymerase II complex or the yeast transcription factor TFIID. These data suggest that transposase represses transcription by preventing assembly of an RNA polymerase II complex at the P-element promoter.
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PMID:Drosophila P-element transposase is a transcriptional repressor in vitro. 184 67

Transcription factors, required for the basal expression of the mouse U6 gene were identified in extracts from HeLa cells. This gene is transcribed at least four times more efficiently than its human counterpart in extracts from mouse or HeLa cells and hence provides an excellent in vitro system for the identification of transcription factors involved in the basal expression of mammalian U6 genes. At least four separate protein components were found to be required in addition to RNA polymerase III for correct synthesis of U6 RNA in vitro. These correspond to: (i) TFIIIB; (ii) a heat labile activity contained in a protein fraction enriched in TFIID; (iii) an, as yet, uncharacterized component contained in the flow-through upon rechromatography on phosphocellulose, and finally; (iv) a protein specifically binding to the mouse U6 gene promoter and transactivating its expression. Transcription factors IIIA and IIIC are not involved in mammalian U6 transcription in vitro. The U6-specific transcription factor has a molecular mass of approximately 90 +/- 10 kDa. It specifically binds to the U6 gene from bp -42 to -78 on the coding and from bp -37 to -79 on the non-coding strand thereby centrally encompassing the PSE motif of the mouse U6 promoter. The binding activity of this protein is correlated with the efficiency with which the U6 gene is transcribed in vitro, thereby indicating a crucial role of the PSE-binding protein for U6 transcription.
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PMID:Identification of transcription factors required for the expression of mammalian U6 genes in vitro. 186 35

Although the human U2 and U6 snRNA genes are transcribed by different RNA polymerases (i.e., RNA polymerases II and III, respectively), their promoters are very similar in structure. Both contain a proximal sequence element (PSE) and an octamer motif-containing enhancer, and these elements are interchangeable between the two promoters. The RNA polymerase III specificity of the U6 promoter is conferred by a single A/T-rich element located around position -25. Mutation of the A/T-rich region converts the U6 promoter into an RNA polymerase II promoter, whereas insertion of the A/T-rich region into the U2 promoter converts that promoter into an RNA polymerase III promoter. We show that this A/T-rich element can be replaced by a number of TATA boxes derived from mRNA promoters transcribed by RNA polymerase II with little effect on RNA polymerase III transcription. Furthermore, the cloned RNA polymerase II transcription factor TFIID both binds to the U6 A/T-rich region and directs accurate RNA polymerase III transcription in vitro. Mutations in the U6 A/T-rich region that convert the U6 promoter into an RNA polymerase II promoter also abolish TFIID binding. Together, these observations suggest that in the human snRNA promoters, unlike in mRNA promoters, binding of TFIID directs the assembly of RNA polymerase III transcription complexes, whereas the lack of TFIID binding results in the assembly of RNA polymerase II snRNA transcription complexes.
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PMID:The cloned RNA polymerase II transcription factor IID selects RNA polymerase III to transcribe the human U6 gene in vitro. 186 50

Highly purified RNA polymerase II was found to be able to weakly recognize the initiator (Inr) present in the adenovirus IVa2 and major late promoters. The association of RNA polymerase II with the Inr was enhanced by the general transcription factors. The Inr was capable of directing the formation of a DNA-protein complex. Transcription competent complexes on the adenovirus major late and IVa2 promoters appear to be formed by alternative pathways mediated through the Inr and/or "TATA" motif. The presence of both motifs, however, is required for efficient transcription utilizing a discrete start site. Complexes formed at either site required transcription factor TFIID, the TATA binding protein. Consistent with this observation, a TFIID requirement was demonstrated for transcription from a mutant adenovirus major late promoter construct lacking a functional TATA motif.
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PMID:The initiator directs the assembly of a transcription factor IID-dependent transcription complex. 189 50

Heat treatment of yeast nuclear extracts abolished the capacity to initiate transcription at RNA polymerase II promoters. Activity was restored by the addition of both recombinant yeast TFIID and partially purified factor b, a yeast fraction shown previously to be required for polymerase II transcription. On the basis of this assay with heat-treated extract, factor b was purified to virtual homogeneity. The factor appears to comprise polypeptides of approximately 85, 75, and 50 kDa, since these three polypeptides co-purify with activity, and since a native mass of about 200 kDa is estimated from glycerol gradient sedimentation and gel filtration.
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PMID:Purification and characterization of yeast RNA polymerase II transcription factor b. 191 15


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