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)

The Sendai virus C proteins, C', C, Y1, and Y2, are a nested set of four independently initiated carboxy-coterminal proteins encoded on the P mRNA from an alternate reading frame. Together the C proteins have been shown to inhibit viral transcription and replication in vivo and in vitro and C' binds the Sendai virus L protein, the presumed catalytic subunit of the viral RNA polymerase. To identify amino acids within the C' protein that are important for binding L, site-directed mutagenesis of the gstC' gene was used to change conserved charged amino acids to alanine, generating nine mutants. Additionally, a tenth natural mutant, gstF170S, was also constructed. Six of the gstC' mutants, primarily in the C-terminal half of C', exhibited a defect in the ability to bind L protein. The mutants were assayed for their effect on in vitro transcription and replication from the antigenomic promoter, and the data suggest in all but one case a direct correlation between the ability of C to bind L and to inhibit these steps in RNA synthesis. Further studies with two nonfusion C mutants showed that this correlation was specifically due to the C' portion, and not the gst portion, of the fusion proteins. To study their individual functions, each of the four C proteins was fused downstream of glutathione S-transferase. The gstC', gstC, gstY1, and gstY1 fusion proteins were all able to bind L protein and to inhibit viral mRNA and (+)-leader RNA synthesis, and antigenome replication in vitro. In addition, the nonfusion C, Y1, and Y2 proteins all inhibited transcription. The inhibition of (+)-leader RNA and mRNA synthesis by wt C proteins (nonfusion) showed nearly identical dose-response curves, suggesting that inhibition occurs early in RNA synthesis.
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PMID:Sendai virus wild-type and mutant C proteins show a direct correlation between L polymerase binding and inhibition of viral RNA synthesis. 1154 62

The replication of human rhinovirus 2 (HRV2), a positive-stranded RNA virus belonging to the Picornaviridae, requires a virus-encoded RNA polymerase. We have expressed in Escherichia coli and purified both a glutathione S-transferase fusion polypeptide and an untagged form of the HRV2 RNA polymerase 3D(pol). Using in vitro assay systems previously described for poliovirus RNA polymerase 3D(pol) (J. B. Flanegan and D. Baltimore, Proc. Natl. Acad. Sci. USA 74:3677-3680, 1977; A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998), we have analyzed the biochemical properties of the two different enzyme preparations. HRV2 3D(pol) is both template and primer dependent, and it catalyzes two types of synthetic reactions in the presence of UTP, Mn(2+), and a poly(A) template. The first consists of an elongation reaction of an oligo(dT)(15) primer into poly(U). The second is a protein-priming reaction in which the enzyme covalently links UMP to the hydroxyl group of tyrosine in the terminal protein VPg, yielding VPgpU. This precursor is elongated first into VPgpUpU and then into VPg-linked poly(U), which is identical to the 5' end of picornavirus minus strands. The two forms of the enzyme are about equally active both in the oligonucleotide elongation and in the VPg-primed reaction. Various synthetic mutant VPgs were tested as substrates in the VPg uridylylation reaction.
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PMID:Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: purification and enzymatic analysis of the RNA-dependent RNA polymerase 3D(pol). 1160 37

Ligand-dependent gene transcription mediated by the nuclear receptors involves the recruitment of transcriptional coactivators to the ligand-binding domain (LBD), which leads to interaction with the basal transcription machinery, and ultimately with RNA polymerase II. Although most of these coactivators are ubiquitously expressed, a tissue-selective coactivator, PGC-1, has recently been characterized. Because PGC-1 and the retinoid X receptors (RXRs) possess an overlapping tissue distribution, we investigated whether PGC-1 is a coactivator for the retinoid X receptors. In a transient transfection assay, PGC-1 augments ligand-stimulated RXR transcription. Furthermore, PGC-1 efficiently enhances the RXR element-driven reporter gene transcription by all three RXR isoforms. An immunoprecipitation assay reveals that PGC-1 and RXRalpha interact in vivo. In addition, a glutathione S-transferase pull-down assay showed that this interaction requires the presence of the LXXLL motif of PGC-1. We demonstrate further, in a mammalian two-hybrid assay, that this physical interaction also requires the presence of the AF-2 region of RXR to interact with the LXXLL motif of PGC-1, which is consistent with our protein-protein interaction results. A time-resolved fluorescence assay shows that a peptide within the NR box of PGC-1 is efficiently recruited by a ligand-bound RXRalpha in vitro. Finally, PGC-1 and TIF2 synergistically enhance ligand-activated RXRalpha transcriptional activity. Taken together, these results indicate that PGC-1 is a bona fide coactivator for RXRalpha.
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PMID:PGC-1 functions as a transcriptional coactivator for the retinoid X receptors. 1171 15

Two open reading frames at the 5'-end of the tomato bushy stunt virus genomic RNA are predicted to encode a 33-kDa (p33) protein and its 92-kDa (p92) readthrough product. From amino acid sequence comparisons with other small single-stranded RNA viruses, these proteins resemble viral components of the replicase-transcriptase complex. To investigate the accumulation of these proteins in the infected cell, two chimeric proteins were produced that expressed either a portion of p33 or the carboxy-terminal "half" of p92 fused with glutathione S-transferase, and polyclonal ascites fluids specific to p33 or p92 were elicited in mice. As expected, the anti-p33 antibody recognized p33 and the p92 readthrough protein, but the anti-p92 antibody was specific for p92. Immunoblot analyses revealed that at an early stage of infection both proteins were associated with the membrane fractions isolated from virus-infected plants, but later in the infection, prior to collapse of the tissues, these proteins were also associated with the cytoplasmic fraction. At all time points in plants and protoplasts p33 was about 20-fold more abundant than p92. A series of mutations derived from an infectious cDNA clone demonstrated that both the p33 and the p92 proteins were required for replication in protoplasts and the ratio of the two proteins was maintained in the replication-competent mutants. The wild-type amber (UAG) and in vitro-generated ochre (UAA) readthrough codon derivatives replicated in protoplasts. However, the tyrosine mutants (UAC or UAU) that were predicted to express only p92 were not viable in protoplasts.
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PMID:The tomato bushy stunt virus replicase proteins are coordinately expressed and membrane associated. 1183 21

In transcriptional regulation, RNA polymerase II (pol II) interacts and forms complexes with a number of protein factors. To isolate and identify the pol II-associated proteins, we constructed a Schizosaccharomyces pombe strain carrying a FLAG tag sequence fused to the rpb3 gene encoding the pol II subunit Rpb3. By immunoaffinity purification with anti-FLAG antibody-resin, a pol II complex containing the Rpb1 subunit with a nonphosphorylated carboxyl-terminal domain (CTD) was isolated. In addition to the pol II subunits, the complex was found to contain three subunits of a transcription factor TFIIF (TFIIF alpha, TFIIF beta, and Tfg3) and TFIIF-interacting CTD-phosphatase Fcp1. The same type of pol II complex could also be purified from an Fcp1-tagged strain. The isolated Fcp1 showed CTD-phosphatase activity in vitro. The fcp1 gene is essential for cell viability. Fcp1 and pol II interacted directly in vitro. Furthermore, by chemical cross-linking, glutathione S-transferase pulldown, and affinity chromatography, the Fcp1-interacting subunit of pol II was identified as Rpb4, which plays regulatory roles in transcription. We also constructed an S. pombe thiamine-dependent rpb4 shut-off system. On repression of rpb4 expression, the cell produced more of the nonphosphorylated form of Rpb1, but the pol II complex isolated with the anti-FLAG antibody contained less Fcp1 and more of the phosphorylated form of Rpb1 with a concomitant reduction in Rpb4. This result indicates the importance of Fcp1-Rpb4 interaction for formation of the Fcp1/TFIIF/pol II complex in vivo.
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PMID:Formation of a carboxy-terminal domain phosphatase (Fcp1)/TFIIF/RNA polymerase II (pol II) complex in Schizosaccharomyces pombe involves direct interaction between Fcp1 and the Rpb4 subunit of pol II. 1183 23

RNA polymerase II (RNAP II) has previously been shown to be required for the pre-mRNA polyadenylation cleavage reaction in vitro. This activity was found to reside solely in the C-terminal domain (CTD) of the enzyme's largest subunit. Using a deletion analysis of glutathione S-transferase-CTD fusion proteins, we searched among the CTD's 52 imperfectly repetitive heptapeptides for the minimal subset that possesses this property. We found that heptads in the vicinity of 30 to 37 contribute modestly more than other sections, but that no specific subsection of the CTD is necessary or sufficient for cleavage. To investigate further the heptad requirements for cleavage, we constructed a series of all-consensus CTDs having 13, 26, 39, and 52 YSPTSPS repeats. We found that the nonconsensus CTD heptads are together responsible for only 20% of the wild-type cleavage activity. Analysis of the all-consensus CTD series revealed that the remaining 80% of the CTD-dependent cleavage activity directly correlates with CTD length, with significant activity requiring approximately 26 or more repeats. These results are consistent with a scaffolding role for the RNAP II CTD in the pre-mRNA cleavage reaction.
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PMID:Requirements of the RNA polymerase II C-terminal domain for reconstituting pre-mRNA 3' cleavage. 1186 48

The general transcription factor TFIID facilitates recruitment of the transcription machinery to gene promoters and regulates initiation of transcription by RNA polymerase II. hTAF(II)130, a component of TFIID, interacts with and serves as a coactivator for multiple transcriptional regulatory proteins, including Sp1 and CREB. A yeast two-hybrid screen has identified an interaction between hTAF(II)130 and heterochromatin protein 1 (HP1), a chromatin-associated protein whose function has been implicated in gene silencing. We find that hTAF(II)130 associates with HP1 in an isoform-specific manner: HP1alpha and HP1gamma bind to hTAF(II)130, but not HP1beta. In addition, we show that endogenous hTAF(II)130 and components of TFIID in HeLa nuclear extracts associate with glutathione S-transferase-HP1alpha and -HP1gamma. hTAF(II)130 possesses a pentapeptide HP1-binding motif, and mutation of the hTAF(II)130 HP1 box compromises the interaction of hTAF(II)130 with HP1. We demonstrate that Gal4-HP1 proteins interfere with hTAF(II)130-mediated activation of transcription. Our results suggest that HP1alpha and HP1gamma associate with hTAF(II)130 to mediate repression of transcription, supporting a new model of transcriptional repression involving a specific interaction between a component of TFIID and chromatin.
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PMID:Isoform-specific interaction of HP1 with human TAFII130. 1195 14

In the present study, we first report two forms of human phosphoserine aminotransferase (PSAT) cDNA (HsPSAT alpha and HsPSAT beta). HsPSAT alpha has a predicted open reading frame comprising 324 amino acids, encoding a 35.2 kDa protein (PSAT alpha), whereas HsPSAT beta consists of an open reading frame comprising 370 amino acids that encodes a 40 kDa protein (PSAT beta). PSAT alpha is identical with PSAT beta, except that it lacks 46 amino acids between Val(290) and Ser(337) of PSAT beta, which is encoded by the entire exon 8 (138 bp). Both PSAT alpha and PSAT beta can functionally rescue the deletion mutation of the Saccharomyces cerevisiae counterpart. Reverse transcriptase-PCR analysis revealed that the expression of PSAT beta mRNA was more dominant when compared with PSAT alpha mRNA in all human cell lines tested. PSAT beta was easily detected in proportion to the level of mRNA; however, PSAT alpha was detected only in K562 and HepG2 cells as a very faint band. The relative enzyme activity of glutathione S-transferase (GST)-PSAT beta expressed in Escherichia coli appeared to be 6.8 times higher than that of GST-PSAT alpha. PSAT mRNA was expressed at high levels (approx. 2.2 kb) in the brain, liver, kidney and pancreas, and very weakly expressed in the thymus, prostate, testis and colon. In U937 cells, the levels of PSAT mRNA and protein appeared to be up-regulated to support proliferation. Accumulation of PSAT mRNA reached a maximum in the S-phase of Jurkat T-cells. These results demonstrate that although two isoforms of human PSAT can be produced by alternative splicing, PSAT beta rather than PSAT alpha is the physiologically functional enzyme required for the phosphorylated pathway, and indicate that the human PSAT gene is regulated depending on tissue specificity as well as cellular proliferation status with a maximum level expression in the S-phase.
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PMID:Characterization of human phosphoserine aminotransferase involved in the phosphorylated pathway of L-serine biosynthesis. 1263

The Tup1-Ssn6 complex is an important corepressor in Saccharomyces cerevisiae that inhibits transcription through interactions with the basal transcription machinery and by remodeling chromatin. In a two-hybrid screen for factors that interact with the Schizosaccharomyces pombe Tup1 ortholog, Tup11, we isolated the pct1+ cDNA. The pct1+ gene encodes an mRNA 5'-triphosphatase, which catalyzes the first step of mRNA capping reactions. Pct1 did not interact with the S. pombe Ssn6 ortholog. In vitro glutathione S-transferase pull-down experiments revealed that Pct1 binds to the WD repeat regions of Tup11 and the functionally redundant Tup12 protein. Similarly, the S. cerevisiae Tup1 protein associates with the mRNA 5'-triphosphatase encoded by the CET1 gene. The highly conserved C-terminal domain of Cet1 interacts with Tup1 in vitro, and Tup1-Ssn6 complexes co-purify with the Cet1 protein, indicating that in vivo interactions also occur between these proteins. Over-expression of CET1 compromised repression of an MFA2-lacZ reporter gene that is subject to Tup1-Ssn6 repression. These genetic and biochemical interactions between Tup1-Ssn6 and Cet1 indicate that the capping enzyme associated with RNA polymerase II is a target of the corepressor complex.
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PMID:Physical and functional interaction of the yeast corepressor Tup1 with mRNA 5'-triphosphatase. 1263 15

The effect of the administration of Thonningia sanguinea (T. S.) on the abundance of individual components of the cytochrome P450 monooxygenase enzyme was examined using Western blotting and competitive reverse-transcriptase-polymerase chain reaction (RT-PCR). We also investigated the time-course of inhibition of T. S. on drug metabolizing enzymes. A single intraperitoneal dose of T. S. extract (5 ml/kg) suppressed CYP, cytochrome b5 and NADPH-CYP reductase activity by 45%, 34% and 22% respectively 24 h after T. S. administration. While T. S. did not have any significant effect on microsomal glutathione S-transferase activity, it inhibited p-nitrophenol hydroxylase (PNPH, CYP2E1) and 7-methoxyresorufin O-demethylase (MROD, CYP 1A2) activities by 37% and 32% respectively at 12 h post-T. S. administration. PNPH, erythromycin N-demethylase (ERDM, CYP 3A1/2) and MROD activities were inhibited by 28-36% 24 h after T. S. injection. Consistent with these observations, the levels of CYP2E1, CYP1A2 and CYP3A2 proteins were also suppressed 24 h post-T. S. administration. While CYP2E1 mRNA was unaffected by T. S. administration, CYP1A2 and CYP3A2 mRNAs were decreased by T. S. Cytosolic glutathione S-transferase activity was increased by 30%, 6 h after T. S injection. These data demonstrate that administration of T. S. differentially affect CYP isoforms in the liver of rats and that T. S. selectively suppresses CYP3A2 and CYP1A2 gene expression.
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PMID:Selective suppression of cytochrome P450 gene expression by the medicinal herb, Thonningia sanguinea in rat liver. 1474 31


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