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)

In the accompanying paper (Horikoshi et al., 1988) the interaction between ATF and the general transcription initiation factors was analyzed by DNAase I footprinting experiments. Here, we use transcription assays to investigate the role of ATF in the assembly of a functional preinitiation complex. Addition of an oligonucleotide containing an ATF binding site inhibits E4 transcription by sequestering ATF. However, following preincubation of the E4 promoter in the nuclear extract, transcription is refractory to inhibition by the ATF oligonucleotide. Formation of this oligonucleotide-refractory complex occurs at an early stage in the overall transcription initiation reaction and is dependent upon ATF and the general transcription factors RNA polymerase II, TFIIB, and TFIID. This latter result suggests that the assembly and maintenance of a functional preinitiation complex involves cooperative interactions among the various transcription factors. The general transcription factor TFIIE, although required for transcriptional activity, is not involved in the assembly of an ATF oligonucleotide-refractory complex. Our results support the possibility that ATF may be required only transiently for assembly of a functional preinitiation complex.
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PMID:Analysis of the role of the transcription factor ATF in the assembly of a functional preinitiation complex. 341 55

Transcription from the major late promoter of adenovirus type 2 DNA (including DNA sequences from 56 nucleotides upstream to 33 nucleotides downstream of the CAP site) was reconstituted with transcription factors purified from HeLa cells. Five components, transcription factors (TF) IIA, -B, -E, -D and RNA polymerase II, were required for accurate initiation of transcription. Kinetic analyses combined with order of addition experiments suggested that TFIIA acted first during the initiation reaction and that this interaction was followed by the action of TFIID. In agreement with these conclusions, both TFIIA and TFIID were required to render a transcription reaction partially resistant to concentrations of Sarkosyl previously shown to inhibit an early step in the formation of a preinitiation complex. Related Sarkosyl studies indicated that the inferred complex was subsequently recognized by RNA polymerase II, which resulted in an increased level of Sarkosyl-resistant transcription (in the presence of TFIIA and TFIID), and that this interaction occurred independently of TFIIB and TFIIE. However, TFIIB and TFIIE were implicated, along with the other factors and RNA polymerase II, in the subsequent formation of a highly stable preinitiation complex, which was inferred from its ability to initiate (with added nucleotides) in the presence of heparin concentrations which blocked unbound factors. The identification of a new transcription factor, which was required only when viral sequences 3' to the major late promoter were part of the transcription unit, is also reported.
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PMID:Factors involved in specific transcription in mammalian RNA polymerase II. Functional analysis of initiation factors IIA and IID and identification of a new factor operating at sequences downstream of the initiation site. 381 43

We describe a new assay system that allows a rapid, direct, and quantitative detection of promoter-dependent in vitro transcription by RNA polymerase II. The template used is a hybrid plasmid containing the adenovirus major late promoter linked to a synthetic 400-base-pair DNA fragment that lacks cytidine residues on the transcribed strand--i.e., generates a transcript with no guanosine residues. In vitro transcriptions are carried out in the absence of GTP or, if the reactions contain GTP, in the presence of RNase T1 and the chain terminator 3'-0-methyl-GTP. Under these conditions the only RNAs that can accumulate, whether from a circular or linearized DNA template, are the 400-nucleotide RNase T1-resistant transcripts resulting from accurate initiation at the major late promoter. Thus, specific transcription can be directly monitored by conventional RNA quantitation methods. Using this fast assay, we show that three basic transcription factors, TFIIB, TFIID, and TFIIE, are absolutely required, in addition to the RNA polymerase II, for specific transcription initiation from the adenovirus major late promoter. Units of activity can be defined for each of these individual components. The applicability of this kind of assay to other systems is discussed.
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PMID:Factors involved in specific transcription by human RNA polymerase II: analysis by a rapid and quantitative in vitro assay. 392 56

A gene-specific transcription factor, called USF, has been partially purified from HeLa cell nuclear extracts. Addition of USF results in a 10 to 20 fold increase in transcription from the adenovirus major late promoter in an in vitro system reconstituted with transcription factors TFIIB, TFIID, TFIIE, and RNA polymerase II. Binding of USF to the promoter inhibits DNAase I cleavages over a 20 base pair region just upstream of the -45 to +35 region shown previously to interact with TFIID. More discriminating footprint analyses using methidiumpropyl-EDTA-Fe(II) as the cleaving agent indicate that USF interacts primarily with the small palindromic DNA sequence GGCCACGTGACC located between positions -63 and -52 of the major late promoter, while TFIID interacts primarily with a 10 base pair DNA region centered on the consensus TATA sequence. Dissociation rate measurements indicate a cooperative interaction between USF and TFIID when simultaneously bound to the promoter DNA.
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PMID:Interaction of a gene-specific transcription factor with the adenovirus major late promoter upstream of the TATA box region. 407 92

Regulation of transcription by RNA polymerase II (pol II) in eukaryotic cells requires both basal and regulatory transcription factors. In this report we have investigated in vitro pol II basal transcription activity during the cell cycle by using nuclear extracts from synchronized HeLa cells. It is shown that pol II basal transcription activity is low in the S and G2 phases and high in early G1 phase and TFIID is the rate limiting component of pol II basal transcription activity during the cell cycle. Further analyses reveal that TFIID exists as a less active form in the S and G2 phases and nuclear extracts from S and G2 phase cells contain a heat-sensitive repressor(s) of TATA box binding protein (TBP). These results suggest that pol II basal transcription activity is regulated by a qualitative change in the TFIID complex, which could involve repression of TBP, during the cell cycle.
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PMID:Cell cycle-dependent regulation of RNA polymerase II basal transcription activity. 747 63

The central RNA polymerase III (Pol III) transcription factor TFIIIB is composed of the TATA-binding protein (TBP), Brf, a protein related to TFIIB, and the product of the newly cloned TFC5 gene. TFIIIB assembles autonomously on the upstream promoter of the yeast U6 snRNA (SNR6) gene in vitro, through the interaction of its TBP subunit with a consensus TATA box located at base pair -30. As both the DNA-binding domain of TBP and the U6 TATA box are nearly twofold symmetrical, we have examined how the binding polarity of TFIIIB is determined. We find that TFIIIB can bind to the U6 promoter in both directions, that TBP is unable to discern the natural polarity of the TATA element and that, as a consequence, the U6 TATA box is functionally symmetrical. A modest preference for TFIIIB binding in the natural direction of the U6 promoter is instead dictated by flanking DNA. Because the assembly of TFIIIB on the yeast U6 gene in vivo occurs via a TFIIIC-dependent mechanism, we investigated the influence of TFIIIC on the binding polarity of TFIIIB. TFIIIC places TFIIIB on the promoter in one direction only; thus, it is TFIIIC that primarily specifies the direction of transcription. Experiments using TFIIIB reconstituted with the altered DNA specificity mutant TBPm3 demonstrate that in the TFIIIB-U6 promoter complex, the carboxy-terminal repeat of TBP contacts the upstream half of the TATA box. This orientation of yeast TBP in Pol III promoter-bound TFIIIB is the same as in Pol II promoter-bound TFIID and in TBP-DNA complexes that have been analyzed by X-ray crystallography.
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PMID:The symmetry of the yeast U6 RNA gene's TATA box and the orientation of the TATA-binding protein in yeast TFIIIB. 749 93

The human TATA-binding protein was expressed in Escherichia coli as a fusion with an N-terminal hexahistidine sequence, partially purified, and used to raise monoclonal antibodies. More than 50 hybridoma clones producing antibodies that reacted in immunoblot assays with HeLa cell TATA-binding protein and its bacterially synthesized derivative were identified. All antibodies examined recognized epitopes within the N-terminal 159 amino acids of the human TATA-binding protein. Further characterization of one monoclonal antibody, MTBP-6, established that it immunoprecipitates both native HeLa cell TATA-binding protein and TATA-binding protein extracted from cells in the presence of 0.5% SDS. Antibody MTBP-6 immunoprecipitates of native, human cell TATA-binding protein contained the TATA-binding protein and additional polypeptides. Immunoprecipitation of both the TATA-binding protein and several additional polypeptides was specifically blocked by bacterially synthesized, hexahistidine-tagged TATA-binding protein, suggesting that MTBP-6 can efficiently recognize the TATA-binding protein in TFIID and other complexes. Consistent with this conclusion, immunoaffinity chromatography on antibody MTBP-6 permitted purification, in active form, of a TATA-binding protein-containing factor required for transcription by RNA polymerase III. These properties suggest that MTBP-6 will be a useful reagent for the purification and characterization of the multiple TBP-containing complexes present in human cells.
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PMID:Purification of an active TATA-binding protein-containing factor using a monoclonal antibody that recognizes the human TATA-binding protein. 750 37

Fractions obtained from HeLa cell extracts were used to study RNA polymerase III-catalyzed transcription from the human 7SK and mouse U6 RNA promoters in vitro. Although both genes depend on two almost identical core promoter elements (TATA box and PSE), different fractions were required. The 7SK promoter revealed full activity with the phosphocellulose B fraction alone. In contrast, efficient transcription from the U6 promoter depended on the additional presence of the C or D fraction. The analysis of the b1 and b2 subfractions (obtained by DEAE-Sephadex chromatography) revealed that for both promoters the b1 and the phosphocellulose D fraction were mutually interchangeable. However, while both fractions were fully equivalent for the 7SK promoter, the U6 promoter revealed an additional requirement for the C fraction in the presence of the b1 fraction. Since the b1 and the D fractions enclose two different complexes of the TATA-binding protein (TBP), B-TFIID and D-TFIID, our results indicate that functionally these two complexes are responsible for the observed differences in transcription of the 7SK and U6 genes.
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PMID:The seemingly identical 7SK and U6 core promoters depend on different transcription factor complexes. 750 70

The RNA polymerase II (Pol II) basal transcription factor TFIID is composed of the TATA box-binding protein (TBP) and several TBP-associated factors (TAFs). TBP is required for Pol II transcription from TATA-containing and TATA-less promoters. TATA-less promoters of mRNA-encoding genes often contain an initiator element at the transcription start site that is sufficient to direct accurate Pol II transcription. Here we address the mechanisms of functional TBP recruitment to the TATA-less initiator-dependent promoter of the mouse terminal deoxynucleotidyl transferase (TdT) gene. We show that the natural TATA-less TdT initiator region is sufficient to promote low levels of specific transcription in vitro and to direct the assembly of a stable preinitiation complex. In contrast to what is observed for several other promoters lacking a consensus TATA element, the TATA-binding activity of TBP is not required for the functional recruitment of TFIID to the natural TATA-less TdT and beta-polymerase promoters. Moreover, a comparison of TBP and highly purified epitope-tagged TFIID reveals that one or several TAFs function independently of distal regulatory elements to mediate initiator-directed (basal) transcription from the natural TATA-less TdT core promoter in crude nuclear extracts. Furthermore, by using a transcription system reconstituted with purified components, we present the first evidence for a basal transcription function of TAFs through the TdT initiator element. Altogether, our results suggest an alternative pathway for TFIID recruitment to initiator-dependent TATA-less class II promoters in which TAF(s) recruit TBP by interacting either directly or indirectly with the initiator region.
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PMID:TATA-binding protein-associated factor(s) in TFIID function through the initiator to direct basal transcription from a TATA-less class II promoter. 751 74

Transcription initiation factor TFIID is a multisubunit complex containing a TATA-box-binding factor (TFIID tau/TBP) and associated polypeptide factors (TAFs) with sizes ranging from M(r) approximately 20,000 to > 200,000. As a result of direct promoter interactions, TFIID nucleates the assembly of RNA polymerase II and other initiation factors into a functional preinitiation complex. Although the native TFIID complex mediates both basal and activator-dependent transcription in reconstituted systems, TBP itself is competent for only basal transcription. Thus, TAFs are essential cofactors for regulated transcription. The complementary DNAs encoding the p230 (M(r) 230,000), p110 and p85 subunits of TFIID have recently been cloned. Here we report the molecular cloning and characterization of the p62, p42, p28 and p22 subunits. These participate in a network of heterogeneous protein-protein interactions within TFIID. Sequence similarities between p62/p42 and the histones H4/H3, respectively, suggest that these subunits have a functional relationship with chromatin.
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PMID:Molecular cloning of Drosophila TFIID subunits. 754 10


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