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)

Hepatitis B virus X protein (HBx) transactivates viral and cellular genes through a wide variety of cis-elements. However, the mechanism is still obscure. Our finding that HBx directly interacts with RNA polymerase II subunit 5 (RPB5), a common subunit of RNA polymerases, implies that HBx directly modulates the function of RNA polymerase (Cheong, J. H., Yi, M., Lin, Y., and Murakami, S. (1995) EMBO J. 14, 142-150). In this context, we examined the possibility that HBx and RPB5 interact with other general transcription factors. HBx and RPB5 specifically bound to transcription factor IIB (TFIIB) in vitro, both of which were detected by either far-Western blotting or the glutathione S-transferase-resin pull-down assay. Delineation of the binding regions of these three proteins revealed that HBx, RPB5, and TFIIB each has two binding regions for the other two proteins. Co-immunoprecipitation using HepG2 cell lysates that express HBx demonstrated trimeric interaction in vivo. Some HBx substitution mutants, which had severely impaired transacting activity, exhibited reduced binding affinity with either TFIIB or RPB5 in a mutually exclusive manner, suggesting that HBx transactivation requires the interactions of both RPB5 and TFIIB. These results indicated that HBx is a novel virus modulator that facilitates transcriptional initiation by stabilizing the association between RNA polymerase and TFIIB through communication with RPB5 and TFIIB.
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PMID:Hepatitis B virus X protein is a transcriptional modulator that communicates with transcription factor IIB and the RNA polymerase II subunit 5. 905 8

Cisplatin is an anticancer agent frequently used as an alternative to the nitrosoureas in brain tumor chemotherapy. We describe the use of a technique of quantitative reverse transcription-polymerase chain reaction (RT-PCR) to examine the damage induced in the glutathione S-transferase (GST)-pi gene by cisplatin and the subsequent repair of this damage in cells of the MGR3 human glioblastoma multiforme cell line. The relationship between cisplatin dose and the extent of damage in the GST-pi gene was determined over cisplatin concentrations (0-10 microM) within the clinically achievable range. Total RNA was purified from control and cisplatin-treated cells, and both the full-length GST-pi cDNA and control 200-bp beta-actin cDNA were amplified by RT-PCR. The cDNA reaction products were electrophoresed, Southern hybridized, and quantitated densitometrically. A decrease in GST-pi mRNA representing damage to the GST-pi gene was observed with increasing cisplatin concentrations, up to a maximum of 75% at 10 microM cisplatin. Repair of the GST-pi gene in cells treated with cisplatin, assessed as recovery of transcriptional activity of the gene, was shown to occur even after 48 hr following drug removal. A potent RNA polymerase II inhibitor, alpha-amanitin, was used to show that the GST-pi mRNA quantitated in this RT-PCR assay resulted from de novo RNA transcription of the GST-pi gene with little contribution from preexisting GST-pi transcripts. The results demonstrate that the GST pi gene, which is actively transcribed and often overexpressed in human glioma cells, is a target for cisplatin, but that the damage to the gene is efficiently repaired in these cells. The RT-PCR assay has the potential for use in the detection of DNA damage induced by genotoxic agents in other actively transcribed genes and for assessing the repair of gene-specific DNA lesions in cells.
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PMID:Detection of DNA damage in transcriptionally active genes by RT-PCR and assessment of repair of cisplatin-induced damage in the glutathione S-transferase-pi gene in human glioblastoma cells. 907 88

As an initial approach to characterizing the molecular structure of the human RNA polymerase II (hRPB), we systematically investigated the protein-protein contacts that the subunits of this enzyme may establish with each other. To this end, we applied a glutathione S-transferase-pulldown assay to extracts from Sf9 insect cells, which were coinfected with all possible combinations of recombinant baculoviruses expressing hRPB subunits, either as untagged polypeptides or as glutathione S-transferase fusion proteins. This is the first comprehensive study of interactions between eukaryotic RNA polymerase subunits; among the 116 combinations of hRPB subunits tested, 56 showed significant to strong interactions, whereas 60 were negative. Within the intricate network of interactions, subunits hRPB3 and hRPB5 play a central role in polymerase organization. These subunits, which are able to homodimerize and to interact, may constitute the nucleation center for polymerase assembly, by providing a large interface to most of the other subunits.
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PMID:Interactions between the human RNA polymerase II subunits. 920 87

An expression vector, pOVEX, has been designed and constructed, combining the advantages of the expression vectors pGEX-3X and pJC2o. The pOVEX vector produces a fusion protein with the 24 kD Onchocerca volvulus glutathione S-transferase (OvGST2) which is easy to purify in one step from bacterial extracts under non-denaturing conditions using glutathione-sepharose chromatography. High yields of fusion protein were produced from this T7 RNA polymerase-dependent expression vector, which were then cleaved by digestion with the factor Xa protease to separate the OVGST2 polypeptide from the expressed protein of interest. This vector will be particularly useful to O. volvulus investigators for the production of O. volvulus antigens for the analyses of host humoral and cellular responses to these proteins and for immunization studies.
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PMID:pOVEX vector: prokaryotic expression and purification of onchocerciasis vaccine candidate antigens as fusion proteins with the 24 kD Onchocerca volvulus glutathione S-transferase. 927 Jul 37

The Sendai virus nested set of C proteins which are expressed in an alternative open reading frame from the P mRNA has been shown to downregulate viral RNA synthesis. Utilizing a glutathione S-transferase (gst) C fusion protein (gstC), we have shown that C protein forms a complex with the L, but not the P, subunit of the viral RNA polymerase. When P, L, and gstC are coexpressed, an oligomer of P, through its interaction with L, is also bound to beads. Since binding of C to L in the P-L complex does not disrupt P binding, the C and P binding sites appear to be different. GstC binding to L occurs only when the proteins are coexpressed in the same cell. The gstC, but not gst, protein inhibits viral transcription in vitro, showing that the fusion protein retains biological function. Pulse-chase experiments of the various complexes show that L protein synthesized alone has a half-life of 1. 2 hr, which is increased 12.5-fold by binding P, but is not significantly increased by binding gstC. Analyses of complex formation with truncations of L protein show that the C-terminal 1333 amino acids of L are not required for binding C. The dose-response curves show that replication of the genomic DI-H RNA is more sensitive to inhibition by C protein than is the synthesis of DI leader RNA, suggesting that the downregulation of RNA synthesis may be more complex than just the inhibition of the initiation of RNA synthesis.
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PMID:The Sendai virus C protein binds the L polymerase protein to inhibit viral RNA synthesis. 928 6

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 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


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