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 analysed the molecular basis for the function of the C/EBP alpha transactivation domain. We have previously found that the three C/EBP alpha transactivation elements (TEs) synergistically activate transcription in mammalian cells. We now report that two of these elements, TE-I and -II, co-operatively mediate in vitro binding of C/EBP alpha to TBP and TFIIB, two essential components of the RNA polymerase II basal transcriptional apparatus. The TBP and TFIIB binding elements of C/EBP alpha coincide, and require amino acid motifs conserved between the activating members of the C/EBP family. These same motifs are necessary for the transcription activation function of TE-I and -II in both yeast and mammalian cells. Our data demonstrate a biochemical basis for the modular buildup of transactivation domains, and indicate that this modularity is conserved in eukaryote evolution. We also show that the same amino acid motifs in a cellular activator can co-operate to mediate contacts between the activator and two distinct basal transcription factors. These results suggest that domains of TBP and TFIIB that interact with activating surfaces are functionally similar and may be structurally related, and support the idea that the same amino acid motifs in an activator carry out multiple functions during the initiation process.
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PMID:CCAAT/enhancer binding protein-alpha amino acid motifs with dual TBP and TFIIB binding ability co-operate to activate transcription in both yeast and mammalian cells. 755 73

Accurate and regulated transcription by RNA polymerase II requires the assembly of an initiation complex involving multiple protein-DNA and protein-protein interactions. A key event is binding of TFIID, a complex consisting of TBP and associated factors (TAFs) to the template DNA. The TAF subunits of TFIID carry out diverse functions critical for transcription, including specific contact with enhancer proteins and binding to core promoter DNA. However, the role of TAFs in RNA polymerase II-mediated transcription initiation and cross talk with other basal factors remains poorly characterized. Here, we report the specific interaction of TAFII250 with RAP74, an essential subunit of the basal transcription factor IIF. Using various in vitro binding assays we have mapped recognition interfaces between TAFII250 and RAP74. In vivo complementation of a temperature-sensitive TAFII250 cell line reveals that the RAP74 interaction is critical for cell viability. Because TFIIF is thought to be responsible for binding and recruiting RNA polymerase II, the ability of TAFII250 to interact selectively with RAP74 is likely to contribute a critical contact for the assembly of an active transcription complex.
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PMID:Human TAFII250 interacts with RAP74: implications for RNA polymerase II initiation. 759 Feb 50

We present homologies between archaeal and eucaryal DNA-dependent RNA polymerase (RNAP) subunits and transcription factors. The sequences of the Sulfolobus acidocaldarius subunits D, E, and N and alignments with eucaryal homologs are presented here. The similarities between archaeal transcription factors and their eucaryal homologs TFIIB and TBP have been established in other laboratories. The archaeal RNAP subunits H, K, and N, respectively, show high sequence similarity to ABC27, ABC23, and ABC10 beta (found in all three eucaryal RNAPs); subunit D, to AC40 (common to polymerase II and polymerase III) and B44 (polymerase II); and subunit L, to AC19 and B12.5. The similarity of subunit D and its eucaryal homologs to bacterial alpha is limited to the "alpha-motif," which is also present in subunit L and its eucaryal homologs. Genes encoding homologs of the related eucaryal RNAP subunits A12.2/B12.6 and also homologs of eucaryal transcription elongation factors of the TFIIS family have been detected in Sulfolobus acidocaldarius and Thermococcus celer. In archaea, the protein is not an RNAP subunit. Together with the sequence similarities between archaeal box A-containing and eucaryal TATA box-containing promoters, this shows that the archaeal and eucaryal transcription systems are truly homologous and that they differ structurally and functionally from the bacterial transcription machinery. In contrast, however, a number of genes for the archaeal transcription apparatus are organized in clusters resembling the clusters of transcription-associated genes in Bacteria.
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PMID:Transcription in archaea: similarity to that in eucarya. 759 27

The Archaea (archaebacteria) constitute a group of prokaryotes that are phylogenetically distinct from Eucarya (eukaryotes) and Bacteria (eubacteria). Although Archaea possess only one RNA polymerase, evidence suggests that their transcriptional apparatus is similar to that of Eucarya. For example, Archaea contain a homolog of the TATA-binding protein which interacts with the TATA-box like A-box sequence upstream of many archaeal genes. Here, we report the cloning of a Sulfolobus shibatae gene that encodes a protein (transcription factor TFB) with striking homology to the eukaryotic basal transcription factor TFIIB. We show by primer extension analysis that transcription of the S. shibatae TFB gene initiates 27 bp downstream from a consensus A-box element. Significantly, S. shibatae TFB contains an N-terminal putative metal-binding region and two imperfect direct repeats--structural features that are well conserved in eukaryotic TFIIBs. This suggests that TFB may perform analogous functions in Archaea and Eucarya. Consistent with this, we demonstrate that S. shibatae TFB promotes the binding of S. shibatae TBP to the A-box element of the Sulfolobus 16S/23S rRNA gene. Finally, we show that S. shibatae TFB is significantly more related to TFB of the archaeon Pyrococcus woesei than it is to eukaryotic TFIIBs. These data suggest that TFB arose in the common archaeal/eukaryotic ancestor and that the lineages leading to P. woesei and S. shibatae separated after the divergence of the archaeal and eukaryotic lines of descent.
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PMID:Molecular cloning of the transcription factor TFIIB homolog from Sulfolobus shibatae. 759 84

We examined the mechanism by which the C-terminal 236 amino acids of the even-skipped protein (region CD) repress transcription. A fusion protein, CDGB, was created that contains region CD fused to the glucocorticoid receptor DNA binding domain. This protein repressed transcription in an in vitro system containing purified fractions of the RNA polymerase II general transcription factors, and repression was dependent upon the presence of high-affinity glucocorticoid receptor binding sites in the promoter. Repression by CDGB was prevented when the promoter DNA was preincubated with TFIID or TBP, whereas preincubation of the template DNA with CDGB prevented TFIID binding. Together, these results strongly imply that CDGB represses transcription by inhibiting TFIID binding, and further experiments suggested a mechanism by which this may occur. Region CD can mediate cooperative interactions between repressor molecules such that molecules bound at the glucocorticoid receptor binding sites stabilize binding of additional CDGB molecules to low-affinity binding sites throughout the basal promoter. Binding to some of these low-affinity sites was shown to contribute to repression. Further experiments suggested that the full-length eve protein also represses transcription by the same mechanism. We speculate that occupancy of secondary sites within the basal promoter by CDGB or the eve protein inhibits subsequent TFIID binding to repress transcription, a mechanism we term cooperative blocking.
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PMID:A domain of the even-skipped protein represses transcription by preventing TFIID binding to a promoter: repression by cooperative blocking. 765 85

We have characterized the ability of various human autoimmune sera to react with RNA polymerase II transcription factors. One serum, which strongly inhibited transcription in a cell-free system, was shown to contain antibodies directed against human TFIIB. The serum did not show reactivity against the other general transcription factors, including human TBP, TFIIE and TFIIF. The inhibition of transcription was directly attributable to depletion of TFIIB activity, as demonstrated by reconstitution of activity with recombinant TFIIB. It has long been recognized that components of the RNA processing machinery are major human autoantigens. The present results show that at least one general transcription factor required for messenger RNA synthesis is an autoantigen as well.
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PMID:Identification of human autoantibodies to transcription factor IIB. 765 39

The transcriptional potential of the hsp70 heat shock gene promoter is established prior to induction by stress. It has been shown previously that the TBP subunit of TFIID is associated with the TATA element and that RNA polymerase II is paused downstream from the transcription start site. In order to identify new interactions involved in establishing this potentiated state, a detailed analysis of the molecular architecture of a single copy of the hsp70 promoter was performed. A suitably marked promoter was stably integrated using P-element-mediated transformation so as to overcome any ambiguity that might be associated with analyzing the five copies of the endogenous gene. Genomic footprinting using DNase I revealed two previously unidentified interactions. First, the GAGA element located at -120 is protected by protein. Secondly, the pattern of DNase I cleavage in the vicinity of the transcription start is found to bear significant similarity to the pattern associated with binding of purified TFIID. Noting that purified GAGA factor and TFIID interact similarly with the hsp70 and H3 promoters, the architecture of the endogenous H3 promoter was analyzed to determine what interactions might be needed to establish a potentiated state containing a paused polymerase. Despite the detection of TFIID and GAGA on the H3 promoter, no paused polymerase is evident. In addition, no proteins appear to interact with the transcription start. These results suggest that the GAGA factor and TFIID are not sufficient to establish a potentiated state containing paused polymerase and that TFIID interactions downstream from the TATA element could be important for pausing.
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PMID:Genomic footprinting of the hsp70 and histone H3 promoters in Drosophila embryos reveals novel protein-DNA interactions. 766 10

TFIIB is an essential component of the machinery that transcribes protein-coding genes. The three-dimensional structure of the human TFIIB core domain (TFIIBc) has been determined using multidimensional heteronuclear magnetic resonance spectroscopy. The molecule consists of two direct repeats that adopt similar alpha-helical folds, conferring pseudo-twofold symmetry. An extensive, central basic surface including an amphipathic alpha helix is critical to the function of TFIIB as a bridge between the TBP-promoter complex and RNA polymerase II and associated general and regulatory transcription factors. Similarities between the TFIIBc and cyclin A folds indicate that elements of the eukaryotic cell cycle control apparatus evolved from more fundamental transcriptional control components, demonstrating a link between the transcription and cell cycle molecular machineries.
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PMID:Solution structure of the C-terminal core domain of human TFIIB: similarity to cyclin A and interaction with TATA-binding protein. 767 13

Levels of mRNA and protein encoded by the TATA-binding protein (tbp) gene are shown to increase dramatically during late spermatogenesis in rodents, culminating in a highly testis-enriched expression pattern. Whereas adult spleen and liver contained roughly 0.7 and 2.3 molecules of TBP mRNA per haploid genome-equivalent, respectively, adult testis contained 80-200 molecules of TBP mRNA per haploid genome-equivalent. Comparison of nuclear and cytoplasmic levels of TBP mRNA in liver and testis suggested that nuclear events (transcription or processing) contribute roughly 12-fold, and cytoplasmic events (mRNA stability) roughly 6-fold, to testis-specific overaccumulation. Levels of nuclear TBP protein in testis cells were, on average, 8- and 11-fold higher than those in liver and spleen cells, respectively. Overexpression of TBP mRNA in testis began about 20 days after birth and reached a plateau around day 40, corresponding to the developmental emergence of haploid cells. Besides TBP, two other components of the general RNA polymerase II machinery, TFIIB and RNA polymerase II, were also overexpressed in testis. By immunostaining, it was found that TBP and RNA polymerase II were particularly rich in round spermatid nuclei. Our results suggest a molecular explanation for how early spermatids are able to accumulate all of the mRNA necessary for the final week of spermiogenesis.
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PMID:High accumulation of components of the RNA polymerase II transcription machinery in rodent spermatids. 767 3

The TATA-box-binding protein TBP exists in the cell complexed with different sets of TBP-associated factors (TAFs). In general, each of these TBP-TAF complexes is dedicated to transcription by a single RNA polymerase. Thus, SL1, TFIID and TFIIIB are required for transcription by polymerases I, II and III, respectively. Here we characterize a fourth TBP-TAF complex called SNAPc. Unlike the other TBP-TAF complexes, SNAPc is implicated in transcription by two types of polymerases; it is required for transcription of both the RNA polymerase II and III small-nuclear RNA genes and binds specifically to the proximal sequence element PSE, a non-TATA-box basal promoter element common to these two types of genes. In addition to TBP, SNAPc is composed of at least three TAFs, SNAP43, SNAP45 and SNAP50. The predicted amino-acid sequence of SNAP43 reveals that it corresponds to a new protein.
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PMID:A TBP-TAF complex required for transcription of human snRNA genes by RNA polymerase II and III. 771 7


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