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)

Chlamydia trachomatis is an obligate intracellular pathogen, long recognized as an agent of blinding eye disease and more recently as a common sexually transmitted infection. Recently, two eukaryotic histone H1-like proteins, designated Hc1 and Hc2, have been identified in Chlamydia. Expression of Hc1 in recombinant Escherichia coli produces chromatin condensation similar to nucleoid condensation observed late in the parasite's own life cycle. In contrast, chromatin decondensation, observed during the early life cycle, accompanies down-regulation and nondetection of Hc1 and Hc2 among internalized organisms. We reasoned that the early upstream open reading frame (EUO) gene product might play a role in Hc1 degradation and nucleoid decondensation since it is expressed very early in the chlamydial life cycle. To explore this possibility, we fused the EUO coding region between amino acids 4 and 177 from C. trachomatis serovar Lz with glutathione S-transferase (GST) and examined the effects of fusion protein on Hc1 in vitro. The purified fusion protein was able to digest Hc1 completely within 1 h at 37 degrees C. However, GST alone exhibited no Hc1-specific proteolytic activity. The chlamydial EUO-GST gene product also cleaves very-lysine-rich calf thymus histone H1 and chicken erythrocyte histone H5 but displays no measurable activity towards core histones H2A, H2B, H3, and H4 or chlamydial RNA polymerase alpha-subunit. This proteolytic activity appears sensitive to the serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) and aspartic protease inhibitor pepstatin but resistant to high temperature and other broad-spectrum protease inhibitors. The proteolytic activity specified by the EUO-GST fusion product selectively digested the C-terminal portion of chlamydial Hc1, the domain involved in DNA binding, while leaving the N terminus intact. At a molar equivalent ratio of 1:1 between Hc1 and DNA, the EUO gene product cleaves Hc1 complexed to DNA and this cleavage appears sufficient to initiate dissociation of DNA-Hc1 complexes. However, at a higher molar equivalent ratio of Hc1/DNA (10:1), there is partial protection conferred upon Hc1 to an extent that prevents dissociation of DNA-Hc1 complexes.
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PMID:The chlamydial EUO gene encodes a histone H1-specific protease. 929 54

Screening of a genomic mouse DNA library with a glutathione S-transferase class mu cDNA probe resulted in the identification of mGSTM5, a novel member of the murine glutathione S-transferase class mu gene family. Here we present the sequence of the promoter region, the exon-intron organization of the gene and the isolation and characterization of its complete cDNA. Conceptual translation of the cDNA sequence revealed that several amino acid positions have been changed in 'invariant' mu class signature sequences in mGSTM5. Reverse transcriptase polymerase chain reaction using gene specific primers revealed that mGSTM5 is uniquely expressed in mouse liver, stomach and small intestine.
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PMID:Identification of a novel murine glutathione S-transferase class mu gene. 948 Aug 67

The rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) RNA-dependent RNA-polymerase (3Dpol) coding region was expressed in Escherichia coli by using a glutathione S-transferase-based vector, which allowed milligram purification of a homogeneous enzyme with an expected molecular mass of about 58 kDa. The recombinant polypeptide exhibited rifampin- and actinomycin D-resistant, poly(A)-dependent poly(U) polymerase. The enzyme also showed RNA polymerase activity in in vitro reactions with synthetic RHDV subgenomic RNA in the presence or absence of an oligo(U) primer. Template-size products were synthesized in the oligo(U)-primed reactions, whereas in the absence of added primer, RNA products up to twice the length of the template were made. The double-length RNA products were double stranded and hybridized to both positive- and negative-sense probes.
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PMID:Expression of enzymatically active rabbit hemorrhagic disease virus RNA-dependent RNA polymerase in Escherichia coli. 952 22

The tumor suppressor gene BRCA1, is a nuclear phosphoprotein which associates with RNA polymerase II holoenzyme. CBP is a component of the holoenzyme. Previously, we have characterized two new BRCA1 splice variants BRCA1a/p110 and BRCA1b/p100. In the present study, the carboxy-terminal domain of transcription factor CBP interacts both in vivo and in vitro with full length BRCA1a and BRCA1b proteins as demonstrated by mammalian two- hybrid assays, co-immunoprecipitation/western blot studies, GST binding assays and histone acetyl transferase (HAT) assays of BRCA1 immunoprecipitates from human breast cancer cells. Our results suggest that one of the mechanisms by which BRCA1 proteins function is through recruitment of CBP associated HAT/FAT (transcription factor acetyl-transferase) activity for acetylation of either themselves or general transcription factors or both to specific promoters resulting in transcriptional activation.
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PMID:BRCA1 splice variants BRCA1a and BRCA1b associate with CBP co-activator. 953 57

RNA polymerase II from the fission yeast Schizosaccharomyces pombe consists of 10 putative subunits. Subunit 3 (Rpb3) is a homologue of prokaryotic alpha subunit, which plays a key role in the assembly of core enzyme subunits. Previously we indicated that Rpb3 also plays an essential role in subunit assembly because it interacts with at least four subunits, two large subunits (Rpb1 and Rpb2) and two medium-sized subunits (Rpb3 and Rpb5) (1), and it constitutes a core subassembly consisting of Rpb2, Rpb3, and Rpb11 (2). Using a synthetic mixture of equimolar amounts of individual subunits, which were all purified from cDNA-expressed Escherichia coli, we found here that Rpb3 also interacts with Rpb11, another alpha homologue. By making a set of Rpb3 deletion derivatives, we carried out mapping of the Rpb5- and Rpb11-contact sites on Rpb3. By far-Western blot and GST pull-down assays, we found that the amino acid sequence between residues 105-263 of Rpb3 is involved in binding Rpb5, and the sequence between residues 105-297 is required for binding Rpb11. Although the Rpb5- and Rpb11-contact sites on Rpb3 overlap each other, both subunits are able to associate with Rpb3 simultaneously. The binding of Rpb5 stabilizes the Rpb3-Rpb11 heterodimer.
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PMID:Location of subunit-subunit contact sites on RNA polymerase II subunit 3 from the fission yeast Schizosaccharomyces pombe. 954 38

Yeast U2 snRNA is transcribed by RNA polymerase II to generate a single non-polyadenylated transcript. A temperature-sensitive yeast strain carrying a disruption in RNT1, the gene encoding a homolog of RNase III, produces 3'-extended U2 that is polyadenylated. The U2 3'-flanking region contains a putative stem-loop that is recognized and cleaved at two sites by recombinant GST-Rnt1 protein in vitro. Removal of sequences comprising the stem-loop structure blocks cleavage in vitro and mimics the effects of Rnt1 depletion in vivo. Strains carrying a U2 gene lacking the Rnt1 cleavage site produce only polyadenylated U2 snRNA, and yet are not impaired in growth or splicing. The results suggest that eukaryotic RNase III may be a general factor in snRNA processing, and demonstrate that polyadenylation is not incompatible with snRNA function in yeast.
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PMID:Depletion of yeast RNase III blocks correct U2 3' end formation and results in polyadenylated but functional U2 snRNA. 964 43

The ZFM1 protein is both a transcriptional repressor and identical to the splicing factor SF1. ZFM1 was shown to interact with and repress transcription from the glycine, glutamine, serine, and threonine-rich transcription activation domain of the sea urchin transcription factor, stage-specific activator protein (SSAP). EWS, a human protein involved in cellular transformation in Ewing's sarcoma tumors, contains an NH2-terminal transcriptional activation domain (NTD) which resembles that of SSAP in both amino acid composition and the ability to drive transcription to levels higher than VP16 in most cell types. Here we report that ZFM1 also interacts with EWS in both two-hybrid assays and glutathione S-transferase pull-down experiments. The region on EWS which interacts with ZFM1 maps to 37 amino acids within its NTD. Overexpression of ZFM1 in HepG2 cells represses the transactivation of reporter gene expression driven by Gal4-EWS-NTD fusion protein and this repression correlates with ZFM1 binding to EWS. Furthermore, two proteins, TLS and hTAFII68, which have extensive homology to EWS, also interact with ZFM1. Recently, it was discovered that EWS/TLS/hTAFII68 are each present in distinct TFIID populations and EWS and hTAFII68 were also found to be associated with the RNA polymerase II holoenzyme. The association of ZFM1 with these proteins implies that one normal cellular function for ZFM1 may be to negatively modulate transcription of target genes coordinated by these cofactors.
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PMID:The transcriptional repressor ZFM1 interacts with and modulates the ability of EWS to activate transcription. 966 Jul 65

A collection of 12 monoclonal antibodies (MAbs) raised against porcine reproductive and respiratory syndrome (PRRS) virus was used to study the antigenic structure of the virus nucleocapsid protein (N). The full-length N gene, encoded by open reading frame 7, was cloned from the Canadian PRRS virus, PA-8. Deletions were introduced into the N gene to produce a series of nine overlapping protein fragments ranging in length from 25 to 112 amino acids. The individual truncated genes were cloned as glutathione S-transferase fusions into a eukaryotic expression vector downstream of the T7 RNA polymerase promoter. HeLa cells infected with recombinant vaccinia virus expressing T7 RNA polymerase were transfected with plasmid DNA encoding the N protein fragments, and the antigenicity of the synthesized proteins was analyzed by immunoprecipitation. Based on the immunoreactivities of the N protein deletion mutants with the panel of N-specific MAbs, five domains of antigenic importance were identified. MAbs SDOW17, SR30, and 5H2.3B12.1C9 each identified independent domains defined by amino acids 30 to 52, 69 to 123, and 37 to 52, respectively. Seven of the MAbs tested specifically recognized the local protein conformation formed in part by the amino acid residues 52 to 69. Furthermore, deletion of 11 amino acids from the carboxy terminus of the nucleocapsid protein disrupted the epitope configuration recognized by all of the conformation-dependent MAbs, suggesting that the carboxy-terminal region plays an important role in maintaining local protein conformation.
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PMID:Antigenic structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus. 980 33

Egr-1 (early-growth response factor-1) is a sequence-specific transcription factor that plays a regulatory role in the expression of many genes important for cell growth, development and the pathogenesis of disease. The transcriptional co-activators CBP (cAMP-response-element-binding-protein-binding protein) and p300 interact with sequence-specific transcription factors as well as components of the basal transcription machinery to facilitate RNA polymerase II recruitment and transcriptional initiation. Here we demonstrate a unique way in which Egr-1 physically and functionally interacts with CBP/p300 to modulate gene transcription. CBP/p300 potentiated Egr-1 mediated expression of 5-lipoxygenase (5-LO) promoter-reporter constructs, and the degree of trans-activation was proportional to the number of Egr-1 consensus binding sites present in wild-type and naturally occurring mutants of the 5-LO promoter. The N- and C-terminal domains of CBP interact with the transcriptional activation domain of Egr-1, as demonstrated by a mammalian two-hybrid assay. Direct protein-protein interactions between CBP/p300 and Egr-1 were demonstrated by glutathione S-transferase fusion-protein binding and co-immunoprecipitation/Western-blot studies. These data suggest that CBP and p300 act as transcriptional co-activators for Egr-1-mediated gene expression and that variations between individuals in such co-activation could serve as a genetic basis for variability in gene expression.
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PMID:cAMP-response-element-binding-protein-binding protein (CBP) and p300 are transcriptional co-activators of early growth response factor-1 (Egr-1). 980 99

Activation of transcription at bacteriophage T4 late promoters and coupling of late transcription to concurrent replication requires a peculiar transcriptional activator, the gp45 sliding clamp of the T4 DNA polymerase. In order to activate transcription, the topologically DNA-linked trimeric gp45 must interact with two T4-encoded RNA polymerase-binding proteins, the gp33 co-activator, and the gp55 late sigma factor. The carboxy termini of gp55 and gp33 share a similar sequence, which has been shown to be required for response of late transcription to activation by gp45. Alanine-scanning mutagenesis of the C terminus of gp55 shows that residues within the short hydrophobic sequence L(D/A)FLYE, are necessary for gp55 to bind to gp45, and to respond maximally to transcriptional activation by gp45. When fused to GST, the peptide SLDFLYE suffices for specific gp45 binding. Thus, it constitutes the main gp55 epitope for gp45 interaction.
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PMID:Activator-sigma interaction: A hydrophobic segment mediates the interaction of a sigma family promoter recognition protein with a sliding clamp transcription activator. 981 12


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