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)

Alveolar soft part sarcoma (ASPS) is an unusual tumor with highly characteristic histopathology and ultrastructure, controversial histogenesis, and enigmatic clinical behavior. Recent cytogenetic studies have identified a recurrent der(17) due to a non-reciprocal t(X;17)(p11.2;q25) in this sarcoma. To define the interval containing the Xp11.2 break, we first performed FISH on ASPS cases using YAC probes for OATL1 (Xp11.23) and OATL2 (Xp11.21), and cosmid probes from the intervening genomic region. This localized the breakpoint to a 160 kb interval. The prime candidate within this previously fully sequenced region was TFE3, a transcription factor gene known to be fused to translocation partners on 1 and X in some papillary renal cell carcinomas. Southern blotting using a TFE3 genomic probe identified non-germline bands in several ASPS cases, consistent with rearrangement and possible fusion of TFE3 with a gene on 17q25. Amplification of the 5' portion of cDNAs containing the 3' portion of TFE3 in two different ASPS cases identified a novel sequence, designated ASPL, fused in-frame to TFE3 exon 4 (type 1 fusion) or exon 3 (type 2 fusion). Reverse transcriptase PCR using a forward primer from ASPL and a TFE3 exon 4 reverse primer detected an ASPL-TFE3 fusion transcript in all ASPS cases (12/12: 9 type 1, 3 type 2), establishing the utility of this assay in the diagnosis of ASPS. Using appropriate primers, the reciprocal fusion transcript, TFE3-ASPL, was detected in only one of 12 cases, consistent with the non-reciprocal nature of the translocation in most cases, and supporting ASPL-TFE3 as its oncogenically significant fusion product. ASPL maps to chromosome 17, is ubiquitously expressed, and matches numerous ESTs (Unigene cluster Hs.84128) but no named genes. The ASPL cDNA open reading frame encodes a predicted protein of 476 amino acids that contains within its carboxy-terminal portion of a UBX-like domain that shows significant similarity to predicted proteins of unknown function in several model organisms. The ASPL-TFE3 fusion replaces the N-terminal portion of TFE3 by the fused ASPL sequences, while retaining the TFE3 DNA-binding domain, implicating transcriptional deregulation in the pathogenesis of this tumor, consistent with the biology of several other translocation-associated sarcomas. Oncogene (2001) 20, 48 - 57.
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PMID:The der(17)t(X;17)(p11;q25) of human alveolar soft part sarcoma fuses the TFE3 transcription factor gene to ASPL, a novel gene at 17q25. 1124 3

Human FCP1 in association with RNAP II reconstitutes a highly specific CTD phosphatase activity and is required for recycling RNA polymerase II (RNAP II) in vitro. Here we demonstrate that targeted recruitment of FCP1 to promoter templates, through fusion to a DNA-binding domain, stimulates transcription. We demonstrate that a short region at the C-terminus of the FCP1 protein is required and sufficient for activation, indicating that neither the N-terminal phosphatase domain nor the BRCT domains are required for transcription activity of DNA-bound FCP1. In addition, we demonstrate that the C-terminus region of FCP1 suffices for efficient binding in vivo to the RAP74 subunit of TFIIF and is also required for the exclusive nuclear localization of the protein. These findings suggest a role for FCP1 as a positive regulator of RNAP II transcription.
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PMID:Transcription activation by targeted recruitment of the RNA polymerase II CTD phosphatase FCP1. 1152 23

The involvement of chromatin structure and organization in transcriptional regulatory pathways has become evident. One unsolved question concerns the molecular mechanisms of chromatin remodeling during in vivo promoter activation. By using a high resolution in vivo analysis we show that when yeast cells are exposed to a regulatory signal the positions of specific nucleosomes change. The system analyzed consists of the basic elements of the Saccharomyces cerevisiae ADH2 promoter, two nucleosomes of which are shown to change the distribution of their positions by few nucleotides in the direction of transcription when the glucose content of the medium is lowered. Such repositioning does not occur in the absence of the ADH2 transcriptional activator Adr1 or in the presence of its DNA-binding domain alone. A construct consisting of the DNA-binding domain plus a 43-amino acid peptide containing the Adr1 activation domain is sufficient to induce the same effect of the full-length protein. Nucleosome repositioning occurs even when the catalytic activity of the RNA polymerase II is impaired, suggesting that the Adr1 activation domain mediates the recruitment of some factor to correctly preset the relevant sequences for the subsequent transcription steps.
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PMID:In vivo changes of nucleosome positioning in the pretranscription state. 1174 18

AfsR is a pleiotropic, global regulator that controls the production of actinorhodin, undecylprodigiosin and calcium-dependent antibiotic in Streptomyces coelicolor A3(2). AfsR, with 993 amino acids, is phosphorylated on serine and threonine residues by a protein serine/threonine kinase AfsK and contains an OmpR-like DNA-binding fold at its N-terminal portion and A- and B-type nucleotide-binding motifs in the middle of the protein. The DNA-binding domain, in-dependently of the nucleotide-binding domain, contributed the binding of AfsR to the upstream region of afsS that locates immediately 3' to afsR and encodes a 63-amino-acid protein. No transcription of afsS in the DeltaafsR background and restoration of afsS transcription by afsR on a plasmid in the same genetic background indicated that afsR served as a transcriptional activator for afsS. Interestingly, the AfsR binding site overlapped the promoter of afsS, as determined by DNase I protection assay and high-resolution S1 nuclease mapping. The nucleotide-binding domain contributed distinct ATPase and GTPase activity. The phosphorylation of AfsR by AfsK greatly enhanced the DNA-binding activity and modulated the ATPase activity. The DNA-binding ability of AfsR was independent of the ATPase activity. However, the ATPase activity was essential for transcriptional activation of afsS, probably because the energy available from ATP hydrolysis is required for the isomerization of the closed complex between AfsR and RNA polymerase to a transcriptionally competent open complex. Thus, AfsR turns out to be a unique transcriptional factor, in that it is modular, in which DNA-binding and ATPase activities are physically separable, and the two functions are modulated by phosphorylation on serine and threonine residues.
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PMID:afsS is a target of AfsR, a transcriptional factor with ATPase activity that globally controls secondary metabolism in Streptomyces coelicolor A3(2). 1195 95

Human upstream binding factor is a nucleolar transcription factor involved in transcription by RNA polymerase I. It contains six HMG box domains; the HMG box is a minor groove DNA-binding domain that has been found in hundreds of proteins with different functions. Among the six HMG box domains in hUBF, the first one can bind to the ribosomal promoter specifically by itself and is essential for the whole protein's DNA binding specificity. Here we report the three-dimensional structure of this first HMG box free in solution determined by multidimensional NMR using (13)C,(15)N-labeled protein. Like the previously determined HMG box structures, hUBF HMG box 1 adopts a twisted L-shape consisting of three alpha-helices: helix 1 (17-30) and helix 2 (38-51) pack onto each other to form the short arm, while helix 3 (57-76) is associated with an extended strand N-terminal to helix 1 and forms the long arm. A cluster of conserved residues, in particular the aromatic residues F21, Y49, and Y60, is important to maintain the fold. The short arm is rigid due to extensive hydrophobic interaction between helix 1 and helix 2, while the long arm is less rigid.
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PMID:Solution structure of the first HMG box domain in human upstream binding factor. 1196 1

Previous studies have established a critical role of both TFIIB and RNA polymerase II (RNAPII) in start site selection in the yeast Saccharomyces cerevisiae. However, it remains unclear how the TFIIB-RNAPII interaction impacts on this process since such an interaction can potentially influence both preinitiation complex (PIC) stability and conformation. In this study, we further investigate the role of TFIIB in start site selection by characterizing our newly generated TFIIB mutants, two of which exhibit a novel upstream shift of start sites in vivo. We took advantage of an artificial recruitment system in which an RNAPII holoenzyme component is covalently linked to a DNA-binding domain for more direct and stable recruitment. We show that TFIIB mutations can exert their effects on start site selection in such an artificial recruitment system even though it has a relaxed requirement for TFIIB. We further show that these TFIIB mutants have normal affinity for RNAPII and do not alter the promoter melting/scanning step. Finally, we show that overexpressing the genetically isolated TFIIB mutant E62K, which has a reduced affinity for RNAPII, can correct its start site selection defect. We discuss a model in which the TFIIB-RNAPII interaction controls the start site selection process by influencing the conformation of PIC prior to or during PIC assembly, as opposed to PIC stability.
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PMID:The role of TFIIB-RNA polymerase II interaction in start site selection in yeast cells. 1213 90

The adenovirus large E1A protein activates transcription from early viral promoters by a mechanism that requires a forty amino acid zinc finger activation domain in E1A conserved region 3 (CR3). Recent results indicate that activation by a Gal4 DNA-binding domain-E1A-CR3 fusion requires an interaction between the E1A-CR3 zinc finger and the Sur2 subunit of the mammalian Mediator (of transcription) complex. Although several host proteins have been shown to bind stably to E1A proteins in adenovirus-infected and -transformed cells, an in vivo interaction with Mediator complex subunits has not been described previously. Using immunoprecipitation and gel filtration analyses of nuclear extracts prepared from HeLa cells infected with adenovirus 5 or mutants that express either large or small E1A specifically and from adenovirus 5-transformed cells, we report here that large E1A, but not small E1A, binds to Mediator complex in vivo. Only approximately 1 to 10% of large E1A is bound to Mediator complex at 18 h postinfection and in transformed cells, probably explaining why Mediator complex subunits were not identified among cellular E1A-binding proteins described earlier. Surprisingly, even though extracted Mediator can quantitatively bind to an E1A-CR3 affinity column, only on the order of 1% of cellular Mediator complex is bound by E1A in vivo. Much of the large E1A bound to Mediator in 293 cells is in a stable complex that includes RNA polymerase II, leading us to suggest that the interaction of E1A-CR3 with Mediator stabilizes the interaction of Mediator with the polymerase. This stabilization of the interaction between Mediator and RNA polymerase II may contribute to the mechanism of activation by E1A-CR3.
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PMID:In vivo association of adenovirus large E1A protein with the human mediator complex in adenovirus-infected and -transformed cells. 1218 2

The NifA protein of Klebsiella pneumoniae is required for transcriptional activation of all nitrogen fixation (nif) operons except the regulatory nifLA genes. At these operons, NifA binds to an upstream activator sequence (UAS), with the consensus TGT-N(10)-ACA, via a C-terminal DNA-binding domain (CTD). Binding of the activator to this upstream enhancer-like sequence allows NifA to interact with RNA polymerase containing the alternative sigma factor, sigma(54). The isolated NifA CTD is monomeric and binds specifically to DNA in vitro as shown by DNase I footprinting. Heteronuclear 3D NMR experiments have been used to assign the signals from the protein backbone. Three alpha-helices have been identified, based on secondary chemical shifts and medium range Halpha(i)-NH(i)( + 1), and NH(i)-NH(i)( + 1) NOEs. On addition of DNA containing a half-site UAS, several changes are observed in the NMR spectra, allowing the identification of residues that are most likely to interact with DNA. These occur in the final two helices of the protein, directly confirming that DNA binding is mediated by a helix-turn-helix motif.
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PMID:Secondary structure and DNA binding by the C-terminal domain of the transcriptional activator NifA from Klebsiella pneumoniae. 1223 81

The cI protein of bacteriophage lambda (lambdacI) activates transcription from promoter P(RM) through an acidic patch on the surface of its DNA-binding domain. Genetic evidence suggests that this acidic patch stimulates transcription from P(RM) through contact with the C-terminal domain (region 4) of the sigma(70) subunit of Escherichia coli RNA polymerase. Here, we identify two basic residues in region 4 of sigma(70) that are critical for lambdacI-mediated activation of transcription from P(RM). On the basis of structural modeling, we propose that one of these sigma(70) residues, K593, facilitates the interaction between lambdacI and region 4 of sigma(70) by inducing a bend in the DNA upstream of the -35 element, whereas the other, R588, interacts directly with a critical acidic residue within the activating patch of lambdacI. Residue R588 of sigma(70) has been shown to play an important role in promoter recognition; our findings suggest that the R588 side-chain has a dual function at P(RM), facilitating the interaction of region 4 with the promoter -35 element and participating directly in the protein-protein interaction with lambdacI.
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PMID:Protein-protein and protein-DNA interactions of sigma70 region 4 involved in transcription activation by lambdacI. 1242 56

A specific t(21;22) chromosomal translocation creates the chimeric EWS/ERG gene in some cases of Ewing's sarcoma. In the resultant EWS/ERG fusion protein, the N-terminal part of the ETS family protein ERG is replaced by the N terminus of the RNA-binding protein EWS. We found that both the EWS/ERG and COL11A2 genes are expressed in the Ewing's sarcoma cell line, CADO-ES1. To investigate a potential role for EWS/ERG in COL11A2 gene expression, we characterized the COL11A2 promoter and tested the ability of wild-type ERG and EWS/ERG sarcoma fusion protein to transactivate COL11A2 promoter using a luciferase assay. We found that expression of EWS/ERG, but not wild-type ERG, transactivated the COL11A2 promoter and that this transactivation required not only the N-terminal region of EWS but also an intact DNA-binding domain from ERG. Electrophoretic mobility shift assay using COL11A2 promoter sequence showed involvement of EWS/ERG in the formation of DNA-protein complexes, and chromatin immunoprecipitation assay revealed direct interaction between COL11A2 promoter and EWS/ERG fusion protein in vivo. EWS/ERG, but not wild-type ERG, bound to RNA polymerase II. Treatment of cells with the histone deacetylase inhibitor trichostatin A enabled ERG to transactivate the COL11A2 promoter, therefore abolishing the differential effects of EWS/ERG and ERG. Taken together, these findings indicate that the COL11A2 gene is regulated both by potential ERG association with a histone deacetylase complex and by direct EWS/ERG recruitment of RNA polymerase II.
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PMID:COL11A2 collagen gene transcription is differentially regulated by EWS/ERG sarcoma fusion protein and wild-type ERG. 1255 43


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