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)

As a result of the t(11;22)(q24;q12) chromosomal translocation characterizing the Ewing family of tumors (ET), the amino terminal portion of EWS, an RNA binding protein of unknown function, is fused to the DNA-binding domain of the ets transcription factor Fli1. The hybrid EWS-Fli1 protein acts as a strong transcriptional activator and, in contrast to wildtype Fli1, is a potent transforming agent. Similar rearrangements involving EWS or the highly homologous TLS with various transcription factors have been found in several types of human tumors. Employing yeast two-hybrid cloning we isolated the seventh largest subunit of human RNA polymerase II (hsRPB7) as a protein that specifically interacts with the amino terminus of EWS. This association was confirmed by in vitro immunocoprecipitation. In nuclear extracts, hsRPB7 was found to copurify with EWS-Fli1 but not with Fli1. Overexpression of recombinant hsRPB7 specifically increased gene activation by EWS-chimeric transcription factors. Replacement of the EWS portion by hsRPB7 in the oncogenic fusion protein restored the transactivating potential of the chimera. Our results suggest that the interaction of the amino terminus of EWS with hsRPB7 contributes to the transactivation function of EWS-Fli1 and, since hsRPB7 has characteristics of a regulatory subunit of RNA polymerase II, may influence promoter selectivity.
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PMID:Oncogenic EWS-Fli1 interacts with hsRPB7, a subunit of human RNA polymerase II. 970 26

Chromosome translocation creates a fusion between the EWSR1 gene and an ETS family gene. The fusion between these two genes is a characteristic feature of Ewing sarcoma. We previously identified a fourth translocation, t(17;22)(q12;q12), in genomic DNA isolated from cells of patients affected with Ewing sarcoma. The discovery of this translocation suggested that there might be a novel EWSR1-ETV4 fusion gene. In the present study, we determined the genomic breakpoint and characterized the chimeric transcript of the EWSR1-ETV4 fusion gene in two t(17;22) Ewing sarcomas. Reverse transcriptase-PCR assay showed an in-frame fusion between the 5'-terminal region of EWSR1 and the 3' end of ETV4 (alias E1AF, PEA3); the chimeric transcript could thus serve as a template for expression of a protein composed of the N-terminal portion of EWSR1 fused to the DNA-binding domain of ETV4. Long PCR and sequence analysis of genomic DNA revealed that either exon 8 or intron 7 of EWSR1 is fused to the same intron of ETV4 in both tumors. Several palindromic oligomer sequences were found close to the breakpoints in both genes. The 159-bp Alu-like sequence was repeated in the breakpoint region of the ETV4 gene. These observations suggest a mechanism of EWSR1-ETV4 gene fusion.
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PMID:The genomic breakpoint and chimeric transcripts in the EWSR1-ETV4/E1AF gene fusion in Ewing sarcoma. 985 36

Activation of transcription at sigma 54-dependent bacterial promoters proceeds via a mechanism that is independent of recruitment of RNA polymerase to the promoter, but is instead totally dependent on activator-driven conformational changes in the promoter-bound RNA polymerase. Understanding of the activation mechanism first requires a detailed description of the interactions taking place in the polymerase holoenzyme and closed complex. The interactions of sigma 54 with core RNA polymerase and promoter DNA were investigated using enzymatic and chemical (hydroxyl radical) protease footprinting of sigma. Regions of sigma were identified that are in direct contact with ligands, or whose conformation changes following ligand binding. A comparison of wild-type sigma and a mutant bearing a deletion of conserved Region I, which is required for response to activator proteins and regulated initiation, revealed differences in the protease sensitivity of free sigma indicating that Region I affects sigma conformation. Comparison of the holoenzyme and closed complex hydroxyl radical footprints revealed that residues of wild-type sigma protected by promoter DNA overlap, to a large extent, the residues of Region I-deleted sigma protected by core polymerase. Region I could thus modify DNA-binding by changing conformation of the DNA-binding domain of sigma 54 in a core polymerase-dependent manner. These differences can account for the modified promoter binding of the Region I-deleted sigma holoenzyme observed by DNA footprinting, and are likely of significance to the Region I-dependent activation of transcription.
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PMID:Region I modifies DNA-binding domain conformation of sigma 54 within the holoenzyme. 987 25

In yeast cells, interaction between a DNA-bound protein and a single component of the RNA polymerase II (poIII) holoenzyme is sufficient to recruit the latter to a promoter and thereby activate gene transcription. Here we review results which have suggested such a simple mechanism for how genes can be turned on. The series of experiments which eventually led to this model was originally instigated by studying gene expression in a yeast strain which carries a point mutation in Gal11, a component of the poIII holoenzyme. In cells containing this mutant protein termed Gall11P, a derivative of the transcriptional activator Gal4 devoid of any classical activating region is turned into a strong activator. This activating function acquired by an otherwise silent DNA-binding protein is solely due to a novel and fortuitous interaction between Gal11P and a fragment of the Gal4 dimerization region generated by the P mutation. The simplest explanation for these results is that tethering Gal11 to DNA recruits the poIII holoenzyme and, consequently, activates gene transcription. Transcription factors that are believed not to be integral part of the poIII holoenzyme but are nevertheless required for this instance of gene activation, e.g. the TATA-binding TFIID complex, may bind DNA cooperatively with the holoenzyme when recruited to a promoter, thus forming a complete poIII preinitiation complex. One prediction of this model is that recruitment of the entire poIII transcription complex and consequent gene activation can be achieved by tethering different components to DNA. Indeed, fusion of a DNA-binding domain to a variety of poIII holoenzyme components and TFIID subunits leads to activation of genes bearing the recognition site for the DNA-binding protein. These results imply that accessory factors, which are required to remove or modify nucleosomes do not need to be directly contacted by activators, but can rather be engaged in the activation process when the poIII complex is recruited to DNA. In fact, recruitment of the poIII holoenzyme suffices to remodel nucleosomes at the PHO5 promoter and presumably at many other promoters. Other events in the process of gene expression following recruitment of the transcription complex, e.g. initiation, promoter clearance, elongation and termination, could unravel as a consequence of the recruitment step and the formation of an active preinitiation complex on DNA. This view does not exclude the possibility that classical activators also act directly on chromatin remodeling and post-recruitment steps to regulate gene expression.
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PMID:Recruitment of the RNA polymerase II holoenzyme and its implications in gene regulation. 989 6

In the early Caenorhabditis elegans embryo, maternally expressed PIE-1 protein is required in germ-line blastomeres to inhibit somatic differentiation, maintain an absence of mRNA transcription, and block phosphorylation of the RNA polymerase II large subunit (Pol II) carboxy-terminal domain (CTD). We have determined that PIE-1 can function as a transcriptional repressor in cell culture assays. By fusing PIE-1 sequences to the yeast GAL4 DNA-binding domain, we have identified a PIE-1 repression domain that appears to inhibit the transcriptional machinery directly. A sequence element that is required for this repressor activity is similar to the Pol II CTD heptapeptide repeat, suggesting that the PIE-1 repression domain might target a protein complex that can bind the CTD. An alteration of this sequence element that blocks repression also impairs the ability of a transgene to rescue a pie-1 mutation, suggesting that this repressor activity may be important for PIE-1 function in vivo.
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PMID:Transcriptional repression by the Caenorhabditis elegans germ-line protein PIE-1. 992 44

Eukaryotic transcriptional activators may function, at least in part, to facilitate the assembly of the RNA polymerase II (pol II) preinitiation complex at the core promoter region through their interaction with a subset of components of the basal transcription machinery. Previous studies have shown that artificial tethering of TATA-binding protein (TBP) to the promoter region is sufficient to stimulate pol II transcription in yeast. To test whether this phenomenon is a general one in eukaryotic pol II transcription, the DNA-binding domain of yeast GAL4 was fused to either Xenopus laevis TBP or TFIIB in order to enable these factors to be efficiently positioned near the transcription start site in a GAL4-binding site-dependent manner. We found that GAL4-xTBP as well as GAL4-xTFIIB directed an increased level of transcription without involvement of the transcriptional activator, suggesting that incorporation of these basal factors into a preinitiation complex (PIC) is a major rate-limiting step accelerated by activator proteins in metazoans. These results show that transcription activation by artificial recruitment of basal transcription machinery can be observed in general among eukaryotic transcription both in vivo and in vitro. Furthermore, failure of recovery of transcription by adding GAL4-xTFIIB after depletion of endogenous TBP with TATA oligo competitor suggests that recruitment of TBP cannot be bypassed for Pol II transcription.
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PMID:Recruitment of TBP or TFIIB to a promoter proximal position leads to stimulation of RNA polymerase II transcription without activator proteins both in vivo and in vitro. 1006 20

Luminescence resonance energy transfer measurements were used to show that binding of E. coli core RNA polymerase induced major changes in interdomain distances in the sigma 70 subunit. The simplest model describing core-induced changes in sigma 70 involves a movement of the conserved region 1 by approximately 20 A and the conserved region 4.2 by approximately 15 A with respect to conserved region 2. The core-induced movement of region 1 (autoinhibition domain) and region 4.2 (DNA-binding domain) provides structural rationale for allosteric regulation of sigma 70 DNA binding properties by the core and suggests that this regulation may not only involve directly the autoinhibition domain of sigma 70 but also could involve a modulation of spacing between DNA-binding domains of sigma 70 induced by binding of core RNAP.
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PMID:Core RNA polymerase from E. coli induces a major change in the domain arrangement of the sigma 70 subunit. 1007 5

TFIIA has initially been identified as a component of transcription initiation complex of RNA polymerase II. Its role in transcription has been controversial. In this paper, we report the characterization and functional analysis of both the Arabidopsis TFIIA large and small subunits. Sequence analysis revealed that Arabidopsis TFIIA is structurally more related to animal than to yeast counterparts. Arabidopsis has at least two genes for the large subunit and one for the small subunit. Both types of genes are constitutively transcribed in various plant organs. The proteins encoded by the cDNA interact each other in yeast 2-hybrid system. Only the N-terminal part of the large subunit is necessary for the interaction with the small subunit. Recombinant Arabidopsis TFIIA polypeptides bind to TBP-DNA complex in gel shift assays. The large subunit of TFIIA can stimulate transcription in yeast and in plant cells when fused to a DNA-binding domain binding to cis sequences upstream of a minimal promoter. This trans-activating activity is localized to a 35 amino acid segment within the evolutionarily unconserved central region.
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PMID:Characterization and functional analysis of Arabidopsis TFIIA reveal that the evolutionarily unconserved region of the large subunit has a transcription activation domain. 1009 79

We have studied the ability of the wt1 tumor suppressor gene product to repress different classes of activation domains previously shown to stimulate the initiation and elongation steps of RNA polymerase II transcription in vivo. Repression assays revealed that WT1 represses all three classes of activation domains: Sp1 and CTF, which stimulate initiation (type I), human immunodeficiency virus type I Tat fused to a DNA-binding domain, which stimulates predominantly elongation (type IIA), and VP16, p53 and E2F1, which stimulate both initiation and elongation (type IIB). WT1 is capable of exerting its repression effect over a significant distance when positioned approximately 1700 bp from the core promoter. Deletion analysis of WT1 indicates that the responsible domain resides within the first 180 N-terminal amino acids of the protein. Nuclear run-ons analyzing the effects of WT1 on initiation of transcription demonstrate inhibition of this process. Our observations imply that WT1 can repress activators that stimulate initiation and/or elongation.
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PMID:The Wilms' tumor suppressor gene (wt1) product represses different functional classes of transcriptional activation domains. 1039 May 30

sigmaN (sigma54) RNA polymerase holoenzyme closed complexes isomerize to open complexes in a reaction requiring nucleoside triphosphate hydrolysis by enhancer binding activator proteins. Here, we characterize Klebsiella pneumoniae sigmaN mutants, altered in the carboxy DNA-binding domain (F354A/F355A, F402A, F403A and F402A/F403A), that fail in activator-dependent transcription. The mutant holoenzymes have altered activator-dependent interactions with promoter sequences that normally become melted. Activator-dependent stable complexes accumulated slowly in vitro (F402A) and to a reduced final level (F403A, F402A/F403A, F354A/F355A). Similar results were obtained in an assay of activator-independent stable complex formation. Premelted templates did not rescue the mutants for stable preinitiation complex formation but did for deleted region I sigmaN, suggesting different defects. The DNA-binding domain substitutions are within sigmaN sequences previously shown to be buried upon formation of the wild-type holoenzyme or closed complex, suggesting that, in the mutants, alteration of the sigmaN-core and sigmaN-DNA interfaces has occurred to change holoenzyme activity. Core-binding assays with the mutant sigmas support this view. Interestingly, an internal deletion form of sigmaN lacking the major core binding determinant was able to assemble into holoenzyme and, although unable to support activator-dependent transcription, formed a stable activator-independent holoenzyme promoter complex on premelted DNA templates.
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PMID:Involvement of the sigmaN DNA-binding domain in open complex formation. 1044 95


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