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)

We have analyzed the core promoter element of the Na+/K(+)-ATPase alpha 1 subunit gene by means of an in vitro transcription system composed of a HeLa nuclear extract. 5'-deletion and 3'-deletion analyses revealed that this gene is specifically transcribed by RNA polymerase II in a manner that is dependent on the upstream regulatory region of the gene (-102 to -61), and that the 3' boundary of the minimal promoter element does not extend beyond +5. Analysis of linker-substitution mutations and point mutations revealed that the TATA-like sequence (-33 to -26) is required for upstream-sequence-dependent transcription whereas linker-substitution mutations and point mutations near +1 did not abolish transcription. The gene was found to be transcribed by RNA polymerase III when phosphocellulose column fractions were assayed. Deletion analysis mapped the minimal RNA-polymerase-III--specific promoter element from -49 to +17. The phosphocellulose 0.3-M-KCl fraction is absolutely required for transcription by RNA polymerase III, while the 0.85-M-KCl fraction represses aberrant transcription from incorrect initiation sites. Analysis of linker-substitution mutations indicated that the TATA-like sequence is required for RNA-polymerase-III--specific transcription. Although point mutations in the 5' half of the TATA-like sequence did not affect transcription, those in the 3' half shifted the transcription initiation site 3 bp upstream. The results suggest the the Na+/K(+)-ATPase alpha 1 subunit gene promoter contains a TATA-like sequence which can direct transcription by RNA polymerase III in vitro. The mechanism of alternative regulation of RNA polymerase II and RNA polymerase III is discussed.
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PMID:Characterization of the core promoter of the Na+/K(+)-ATPase alpha 1 subunit gene. Elements required for transcription by RNA polymerase II and RNA polymerase III in vitro. 864 83

Transcription factor IIH (TFIIH) is a multisubunit complex required for transcription and for DNA nucleotide excision repair. TFIIH possesses three enzymatic activities: (i) an ATP-dependent DNA helicase, (ii) a DNA-dependent ATPase, and (iii) a kinase with specificity for the carboxyl-terminal domain of RNA polymerase II. The kinase activity was recently identified as the cdk (cyclin-dependent kinase) activating kinase, CAK, composed of cdk7, cyclin H, and MAT-1. Here we report the isolation and characterization of three distinct CAK-containing complexes from HeLa nuclear extracts: CAK, a novel CAK-ERCC2 complex, and TFIIH. CAK-ERCC2 can efficiently associate with core-TFIIH to reconstitute holo-TFIIH transcription activity. We present evidence proposing a critical role for ERCC2 in mediating the association of CAK with core TFIIH subunits.
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PMID:Human cyclin-dependent kinase-activating kinase exists in three distinct complexes. 869 42

Four different plasma membrane Ca(2+)-ATPase (PMCA) genes and three sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) genes have been previously cloned and characterized. In this study we have investigated the expression of the mRNA encoding the various PMCA and SERCA proteins in fetal and adult human heart and placenta by the reverse-transcriptase-polymerase-chain-reaction (RT-PCR) and cDNA cloning. We have found that PMCA1 and PMCA4 genes were expressed in 8-, 12- and 20-week fetal heart and in adult heart. PMCA2 gene was expressed at low levels in adult heart but was not detected in fetal heart. PMCA3 mRNA was not detected in the heart nor placenta. In contrast, the mRNA encoding SERCA2a, SERCA2b and SERCA3 were expressed in all cardiac developmental stages. Multiple alternatively spliced mRNA transcripts which differ at splice site A and B/C of the PMCA1, PMCA2 and PMCA4 genes were detected in the human heart. Interestingly, a novel tissue specific variant of the PMCA4 gene was detected in both fetal and adult human heart but not in placenta that accounts for about 30% of the total PMCA4 mRNA variant expression. DNA sequence analysis of this novel variant revealed that it corresponds to the equivalent of the PMCA1d variant and accordingly we have named it PMCA4d. We cloned and sequenced eight cDNA inserts encoding for the PMCA1 and PMCA4 variants from a fetal human heart cDNA library confirming that these are the two main PMCA genes expressed in cardiac muscle.
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PMID:Analysis of mRNA expression and cloning of a novel plasma membrane Ca(2+)-ATPase splice variant in human heart. 870 Jan 62

Cells from Cockayne's syndrome (CS) patients are sensitive to ultraviolet light and defective in preferential repair of the transcribed DNA strand. CS patients suffer from complex clinical symptoms, including severe growth retardation, neurological degeneration, mental retardation, and cachexia. Two CS complementation groups, CSA and CSB, have been identified so far. RAD26 encodes the yeast counterpart of the CSB gene. Here, we purify Rad26 protein to near homogeneity from yeast cells and show that it is a DNA-dependent ATPase. In contrast to the Mfd protein that functions in transcription-coupled repair in Escherichia coli, and which is a weak and DNA independent ATPase, Rad26 is a much more active ATPase, with a strict dependence on DNA. The possible role of Rad26 ATPase in the displacement of stalled RNA polymerase II from the site of the DNA lesion and in the subsequent recruitment of a DNA repair component is discussed.
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PMID:RAD26, the yeast homolog of human Cockayne's syndrome group B gene, encodes a DNA-dependent ATPase. 870 68

We have investigated the over-production of seven membrane proteins in an Escherichia coli-bacteriophage T7 RNA polymerase expression system. In all seven cases, when expression of the target membrane protein was induced, most of the BL21(DE3) host cells died. Similar effects were also observed with expression vectors for ten globular proteins. Therefore, protein over-production in this expression system is either limited or prevented by bacterial cell death. From the few survivors of BL21(DE3) expressing the oxoglutarate-malate carrier protein from mitochondrial membranes, a mutant host C41(DE3) was selected that grew to high saturation cell density, and produced the protein as inclusion bodies at an elevated level without toxic effect. Some proteins that were expressed poorly in BL21(DE3), and others where the toxicity of the expression plasmids prevented transformation into this host, were also over-produced successfully in C41(DE3). The examples include globular proteins as well as membrane proteins, and therefore, strain C41(DE3) is generally superior to BL21(DE3) as a host for protein over-expression. However, the toxicity of over-expression of some of the membrane proteins persisted partially in strain C41(DE3). Therefore, a double mutant host C43(DE3) was selected from C41(DE3) cells containing the expression plasmid for subunit b of bacterial F-ATPase. In strain C43(DE3), both subunits b and c of the F-ATPase, an alanine-H(+) symporter, and the ADP/ATP and the phosphate carriers from mitochondria were all over-produced. The transcription of the gene for the OGCP and subunit b was lower in C41(DE3) and C43(DE3), respectively, than in BL21(DE3). In C43(DE3), the onset of transcription of the gene for subunit b was delayed after induction, and the over-produced protein was incorporated into the membrane. The procedure used for selection of C41(DE3) and C43(DE3) could be employed to tailor expression hosts in order to overcome other toxic effects associated with over-expression.
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PMID:Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. 875 92

We previously found that nusD-type mutations in Escherichia coli transcription termination factor Rho enhance in vitro transcription termination at four points within the lambdacro gene. Here we show that the early termination points are part of one Rho-dependent termination site, tRE, with properties like those of previously characterized Rho-dependent sites lamda tR1 and trpt'. The early termination points are all RNA polymerase pause sites, and by deletion analysis and oligonucleotide blocking experiments, a common 5' Rho entry site for the early termination points (rutE) is identified. We show that both Rho026 and Rho+ can use rutE as an entry point for termination, but that Rho026 is more efficient in releasing the nascent RNA at tRE. The RNA-dependent ATPase activities of wild-type and mutant Rhos are similar, as are their abilities to bind free RNA and to use (rC)10 oligomers for ATPase activation. We therefore suggest that Rho-RNA polymerase interactions that define the site of RNA 3' end formation are altered in NusD Rho mutants. NusD Rho mutants are less dependent on, but still responsive to, the transcription termination factor NusG. However, addition of NusG to in vitro termination assays allows Rho+ to terminate more efficiently at tRE. These results suggest that NusG aids in the 3' end formation process. The decreased dependence on NusG for termination by the mutant Rhos in vitro provides an explanation for poorer lambda growth in rho(nusD) cells by interference with lamdaN-mediated antitermination at Rho-dependent sites.
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PMID:The mechanism of early transcription termination by Rho026. 875 98

To investigate the regulation of the Na,K-ATPase, we have studied the expression of the Na,K-ATPase polypeptides in several mammalian cell lines using the vaccinia virus/T7 RNA polymerase expression system. Infection of several fibroblast-like cell lines with viral recombinants containing the Na,K-ATPase alpha and beta isoforms, the glucose transporters, GLUT 1 and GLUT 4, or the capsid protein of the Sindbis virus all result in the production of the appropriate protein products. However, all epithelial cell lines tested fail to synthesize the Na,K-ATPase viral recombinants, yet they efficiently express the other virally directed polypeptides. While Madin-Darby canine kidney (MDCK) epithelial cells infected with the Na,K-ATPase alpha1 or beta1 recombinant viruses produce both mRNAs, the messages are inefficiently translated. Furthermore, the RNA from infected MDCK cells does not direct the in vitro synthesis of the beta1 polypeptide, whereas the message from infected fibroblast-like BSC 40 cells is efficiently translated both in vivo and in vitro. Moreover, the synthesis of the H,K-ATPase alpha subunit is also limited in MDCK cells, although the H,K-ATPase beta subunit is efficiently expressed. Expression of chimeras constructed between the Na+ pump beta1 isoform and the H,K-ATPase beta subunit indicates that sequences in the 5' coding region of the beta1 message have an inhibitory effect; however, the stringent translational regulation of the beta1 isoform in MDCK cells requires the 5' and 3' regions of the coding sequence. The ability of the polarized cell lines to limit the synthesis of the Na+ pump polypeptides while expressing other vaccinia recombinants at high levels suggests that the polarized cells possess a stringent mechanism for the specific translational regulation of a select set of messages.
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PMID:Translational regulation of Na,K-ATPase alpha1 and beta1 polypeptide expression in epithelial cells. 879 17

The interaction of Rho and the antibiotic bicyclomycin was probed using in vitro transcription termination reactions, poly(C) binding assays, limited tryptic digestions, and the bicyclomycin inhibition kinetics of ATPase activity in the presence of poly(dC) and ribo(C)10. The approximate I50 value for the bicyclomycin inhibition of transcription termination at Rho-dependent sites within a modified trp operon template was 5 microM. At antibiotic concentrations near the I50 value, bicyclomycin inhibition of Rho-dependent transcripts was accompanied by the appearance of a new set of transcripts whose size was midway between the Rho-dependent transcripts and the readthrough transcripts. Bicyclomycin did not inhibit poly(C) binding to Rho. In the presence of poly(dC), bicyclomycin showed a reversible mixed inhibition of the ribo(C)10-stimulated ATPase activity. The extrapolated Ki for bicyclomycin was 2.8 microM without ribo(C)10 and increased to 26 microM in the presence of ribo(C)10. Correspondingly, the Km(app) for ribo(C)10 without bicyclomycin was 0.8 microM and with bicyclomycin was 5 microM at infinite inhibitor concentration. The data suggested that the antibiotic binds to Rho, influencing the secondary RNA binding (tracking) site on Rho and slows the tracking of Rho toward the bound RNA polymerase.
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PMID:The antibiotic bicyclomycin affects the secondary RNA binding site of Escherichia coli transcription termination factor Rho. 881 Mar 2

Transcription by RNA polymerase utilizing the alternative sigma factor beta 54 is regulated by a distinct class of positive activators designated the sigma 54-dependent family. The activities of these regulators are themselves modulated in response to a wide variety of environmental signals. Factors that modulate the expression or the activity of the regulatory protein in response to chemical and metabolic changes are ultimately responsible for determining the level of expression of sigma 54-dependent genes and hence the diverse bacterial functions that they encode. Many members of the sigma 54-dependent family are part of two-component sensor-response systems. This MicroReview emphasizes recent data concerning the activities of a distinct subgroup of the sigma 54-dependent regulators that directly sense and respond with transcriptional activation to the presence of small effector molecules in their environment. The functional consequences of effector activation in terms of regulation of the enzymatic (ATPase) activity of these transcriptional activators and interdomain interactions are discussed.
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PMID:Signal sensing by sigma 54-dependent regulators: derepression as a control mechanism. 883 Feb 33

Human P-glycoprotein (Pgp) is a 170-kDa plasma membrane protein that confers multidrug resistance to otherwise sensitive cells. A mutation in Pgp, G185-->V, originally identified as a spontaneous mutation, was shown previously to alter the drug resistance profiles in cell lines that are stably transfected with the mutant MDR1 cDNA and selected with cytotoxic agents. To understand the mechanism by which the V185 mutation leads to an altered drug resistance profile, we used a transient expression system that eliminates the need for drug selection to attain high expression levels and allows for the rapid characterization of many aspects of Pgp function and biosynthesis. The mutant and wild-type proteins were expressed at similar levels after 24-48 h in human osteosarcoma (HOS) cells by infection with a recombinant vaccinia virus encoding T7 RNA polymerase and simultaneous transfection with a plasmid containing MDR1 cDNA controlled by the T7 promoter. For both mutant and wild-type proteins, photolabeling with [3H]azidopine and [125I]iodoarylazidoprazosin, drug-stimulated ATPase activity, efflux of rhodamine 123, and accumulation of radiolabeled vinblastine and colchicine were evaluated. In crude membrane preparations from HOS cells, a higher level of basal Pgp-ATPase activity was observed for the V185 variant than for the wild-type, suggesting partial uncoupling of drug-dependent ATP hydrolysis by the mutant. Several compounds, including verapamil, nicardipine, tetraphenylphosphonium, and prazosin, stimulated ATPase activities of both the wild-type and mutant similarly, whereas cyclosporin A inhibited the ATPase activity of the mutant more efficiently than that of the wild-type. This latter observation explains the enhanced potency of cyclosporin A as an inhibitor of the mutant Pgp. No differences were seen in verapamil-inhibited rhodamine 123 efflux, but the rate of accumulation was slower for colchicine and faster for vinblastine in cells expressing the mutant protein, as compared with those expressing wild-type Pgp. We conclude that the G185-->V mutation confers pleiotropic alterations on Pgp, including an altered basal ATPase activity and altered interaction with substrates and the inhibitor cyclosporin A.
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PMID:Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system. 889 56


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