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)

Cultures of the rat skeletal muscle myoblast cell line, L6, were treated with the mutagen ethylmethanesulfonate and grown in the presence of alpha-amanitin, an inhibitor of RNA polymerase II in vitro. One clonal cell line, Ama102, resistant tc the cytotoxic action of 2 mu-g/ml of alpha-amanitin was isolated and extensively characterized. Ama102 cells were about 30-fold more resistant to alpha-amanitin than their Ama+ parent cells based on a comparison of the concentration of alpha-amanitin required to reduce their plating efficiencies to similar extents. The RNA polymerase activities from Ama+ and Ama102 cells were solubilized and separated by DEAE-Sephadex chromatography. Whereas all of the Ama+ RNA polymerase II activity was inhibited by 0.1 mu-g/ml of alpha-amanitin, about 30% of the activity in the Ama102 RNA polymerase II peak was resistant to this concentration of alpha-amanitin and was inhibited only by much higher concentrations (25 mu-g/ml) of alpha-amanitin. This alpha-amanitin-resistant activity in Ama102 cells was identified as a bona fide RNA polymerase II by its chromatographic behavior on DEAE-Sephadex, salt optimum, preference for denatured DNA as template, insensitivity to inhibition by potassium phosphate, thermal inactivation kinetics, and inactivation by anti-RNA polymerase II antiserum. Both RNA polymerase IIa and IIb from Ama102 cells exhibited the partial alpha-amanitin resistance, as did this activity when purified further on phosphocellusose. Unlike the parental Ama+ cells, Ama102 cells neither fused at confluence nor showed an increase in the specific activity of creatine kinase. The altered sensitivity of the Ama102 RNA polymerase II to alpha-amanitin appears to account for the drug-resistant phenotype of these cells.
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PMID:Isolation and characterization of an alpha-amanitin-resistant rat myoblast mutant cell line possessing alpha-amanitin-resistant RNA polymerase II. 16 92

DNA alpha-polymerase has been partially purified from nuclei of cultured chic, fibroblasts and separated on phosphocellulose columns into two distinct activities designated DNA polymerases alpha(a) and alpha(b), respectively. The enzyme preparations were devoid of activities of DNA beta,gamma-polymerases terminal deoxyribonucleoside transferase, DNase, DNA-dependent RNA polymerase, and phosphatase. DNA polymerases alpha(a) and alpha(b) both having molecular weights of 160 000, constitute 35-50 and 65-50%, respectively, of the activity of alpha-polymerase in the nucleus. These enzymes differ in their requirements for maximal activity, their relative ability to copy oligo(dG)-poly(dC), their response to ribonucleoside triphosphates, and their kinetics of heat inactivation. When the properties of alpha polymerases derived from early or late passage cultures have been compared, no difference could be detected as a function of cell age in the specific activities of the polymerases in crude cell extracts, their chromatographic behavior on diethylaminoethylcellulose and phosphocellulose columns, and their relative abilities to utilize single deoxyribonucleoside triphosphates with activated DNA template. On the other hand, both enzymes become partially heat labile in aging cells. Also, the activity of DNA polymerase alpha(a) from young cells was stimulated by 2--10 mM adenosine or cytidine triphosphates, whereas the same enzyme from old cultures was inhibited by these agents. Conversely, these ribonucleoside triphosphates inhibited the activity of polymerase alpha(b) in young cells but slightly stimulated this enzyme derived from senescent fibroblasts. In addition, the relative ability of DNA polymerase alpha(a) to copy oligo(dG)-poly(dC) decreased in aged cells, whereas that of DNA polymerase alpha(b) increased. We have also observed significant differences in the effects of potassium chloride and N-ethylmaleimide on the activity of DNA polymerase alpha(a) from old cells as compared to young cells. These age-related alterations in the properties of the two avian DNA polymerases may reflect structural or conformational changes in these enzymes.
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PMID:Altered nuclear deoxyribonucleic acid alpha-polymerases in senescent cultured chick embryo fibroblasts. 98 31

The lytic-lysogenic switch in transposable, Mu-like bacteriophage D108 is governed by two divergent and slightly overlapping transcription units originating from the Pe and Pc promoters. DNase I footprinting and in vivo mutational analysis suggest that lysogeny is maintained by c-repressor occupancy of the O2 operator, which precludes RNA polymerase from binding to Pe. Lytic development is controlled by the Ner repressor, which binds to a site symmetrically situated between the converging promoters and, in the absence of other factors, prevents RNA polymerase from binding to either Pc or Pe. DNase I protection and potassium permanganate hypersensitivity in the presence of integration host factor (IHF), which binds and alters the DNA structure upstream of Pe, revealed that RNA polymerase was able to bind Pe irrespective of the Ner.DNA-bound complex, and partially unwind the Pe "-10 region." Ner repression of Pe transcription in vitro was significantly more effective in the absence of IHF. Using a cloned D108 early region-lacZ fusion in IHF-deficient and -proficient backgrounds, we also demonstrate this host factor's affect on ner-repressed Pe in vivo, and generate a system for isolating mutants in the regulatory genes and sites controlling this genetic switch. D108 lytic growth is proposed to occur through IHF-mediated activation of the phage Ner-repressed early operon.
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PMID:Integration host factor activates the Ner-repressed early promoter of transposable Mu-like phage D108. 132 51

In vivo dimethyl sulfate footprinting of the Bacillus subtilis glnRA regulatory region under repressing and derepressing conditions demonstrated that the GlnR protein, encoded by glnR, interacts with two sites situated within and adjacent to the glnRA promoter. One site, glnRAo1, between positions -40 and -60 relative to the start point of transcription, is a 21-bp symmetrical element that has been identified as essential for glnRA regulation (H. J. Schreier, C. A. Rostkowski, J. F. Nomellini, and K. D. Hirschi, J. Mol. Biol. 220:241-253, 1991). The second site, glnRAo2, is a quasisymmetrical element having partial homology to glnRAo1 and is located within the promoter between positions -17 and -37. The symmetry and extent of modifications observed for each site during repression and derepression indicated that GlnR interacts with the glnRA regulatory region by binding to both sites in approximately the same manner. Experiments using potassium permanganate to probe open complex formation by RNA polymerase demonstrated that transcriptional initiation is inhibited by GlnR. Furthermore, distortion of the DNA helix within glnRAo2 occurred upon GlnR binding. While glutamine synthetase, encoded by glnA, has been implicated in controlling glnRA expression, analyses with dimethyl sulfate and potassium permanganate ruled out a role for glutamine synthetase in directly influencing transcription by binding to operator and promoter regions. Our results suggested that inhibition of transcription from the glnRA promoter involves GlnR occupancy at both glnRAo1 and glnRAo2. In addition, modification of bases within the glnRAo2 operator indicated that control of glnRA expression under nitrogen-limiting (derepressing) conditions included the involvement of a factor(s) other than GlnR.
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PMID:Interaction of the Bacillus subtilis glnRA repressor with operator and promoter sequences in vivo. 134 63

Escherichia coli RNA polymerase can terminate transcription efficiently at rho-independent terminators in a purified transcription system in the absence of accessory factors. This process of "intrinsic termination" involves direct recognition of the terminator by the core RNA polymerase, and provides an important model system for the study of the molecular interactions involved in the switch between elongation and termination. We have analyzed the intrinsic termination efficiency (%T) of 13 rho-independent terminators, under a variety of in vitro reaction conditions. Although all of these sites share the general sequence features of typical rho-independent terminators, we find a wide range of %T (2% to 90%) for the different sites under our standard transcription conditions. While %T for a particular site is characteristic of that site, the efficiency can be altered considerably by the nature and concentration of salts in the reaction, by alteration of the concentrations of the nucleoside triphosphate substrates, or by transcription from supercoiled rather than linear templates. Surprisingly, different conditions can alter %T to a different extent for different terminators. For neutral salts such as potassium chloride or potassium glutamate, changes in the range from 0.1 to 1 M affect %T for different terminators in a distinct manner, depending on the terminator and the anion involved. At some sites, %T is greatly increased by Cl- concentrations up to 1 M, while at other sites %T is reduced or unaffected by these conditions. At some sites K+ concentrations up to 1 M give a modest increase in %T, while at other sites %T is slightly reduced under the same conditions. Thus the actual values of %T, as well as the order of terminator sites ranked according to %T, can be altered greatly according to the choice of reaction conditions. Reduction of the Mg2+ concentration below 1 mM has a dramatic and quite different effect, enhancing termination to approximately 100% for all terminators tested. Transcription of supercoiled DNA templates gives somewhat reduced %T as compared with linear DNA templates. However, the effect is no greater than twofold. Our results are not consistent with those expected for models in which %T is determined by the differential stability of DNA, RNA and hybrid duplex structures at the melted region in the transcription complex. Thus, the Cl anion does not affect the stability of nucleic acid duplexes even at 1 M concentrations, but can enhance termination tenfold. Also, the alterations of monovalent cation concentration that affect %T are not expected to have a differential effect on Tm for DNA, RNA and hybrid duplexes.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Parameters affecting transcription termination by Escherichia coli RNA polymerase. I. Analysis of 13 rho-independent terminators. 137 65

A conditional block to transcriptional elongation is an important mechanism for regulating c-myc gene expression. This elongation block within the first c-myc exon was defined originally in mammalian cells by nuclear run-on transcription analyses. Subsequent oocyte injection and in vitro transcription analyses suggested that sequences near the end of the first c-myc exon are sites of attenuation and/or premature termination. We report here that the mapping of single stranded DNA in vivo with potassium permanganate (KMnO4) and nuclear run-on transcription assays reveal that polymerase is paused near position +30 relative to the major c-myc transcription initiation site. Deletion of 350 bp, including the sites of 3'-end formation and intrinsic termination defined in oocyte injection and in vitro transcription assays does not affect-the pausing of polymerase in the promoter-proximal region. In addition, sequences upstream of +47 are sufficient to confer the promoter-proximal pausing of polymerases and to generate the polarity of transcription farther downstream. Thus, the promoter-proximal pausing of RNA polymerase II complexes accounts for the block to elongation within the c-myc gene in mammalian cells. We speculate that modification of polymerase complexes at the promoter-proximal pause site may determine whether polymerases can read through intrinsic sites of termination farther downstream.
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PMID:The block to transcriptional elongation within the human c-myc gene is determined in the promoter-proximal region. 142 80

Yeast RNA viruses include L-A (and its toxin-encoding satellites M1, M2, ...) and L-BC dsRNA viruses and the single-stranded replicons 20S RNA and 23S RNA. L-A has a single-segment 4.6-kb linear genome encoding a major coat protein (gag) and its RNA-dependent RNA polymerase (pol), the latter expressed as a gag-pol fusion protein formed by a -1 ribosomal frameshift. In vitro replication, transcription, and binding systems for L-A have been used to define cis sites necessary for packaging and replication of viral RNA. Cellular functions that promote viral replication include the MAK3-encoded N-acetyltransferase whose modification of the gag N terminus is necessary for L-A virus assembly. The toxins encoded by the M satellite RNAs are processed by enzymes (KEX1 and KEX2, for killer expression) whose study led to discovery of mammalian hormone-processing enzymes. 20S RNA is an apparently naked circular RNA replicon (with a dsRNA form called W) encoding a RNA polymerase-like molecule. Its copy number is induced 10,000-fold in 1% potassium acetate, and it is subject to the same SKI antiviral system that represses L-A, L-BC, and M dsRNA copy number.
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PMID:Double-stranded and single-stranded RNA viruses of Saccharomyces cerevisiae. 144 59

Escherichia coli MelR protein binds to two sites located upstream of the melAB transcription start site. Although both sites are required for optimal melibiose-dependent expression from the melAB promoter, some MelR-dependent expression is found if the upstream site is deleted or if the spacing between the two sites is altered. Gel retardation assays have been exploited to study MelR binding to a DNA fragment carrying just the upstream site. Methylation interference analysis was used to identify one guanine (at -104) which is important for MelR binding. Mutational analysis confirmed the importance of this base and revealed a second position (at -110) where mutations interfere with melAB promoter activity. Experiments using potassium permanganate as a probe suggested that the DNA sequence around -110 adopts a distorted conformation. We propose that the mutation at -104 alters MelR binding by interfering with a direct contact, whereas the mutation at -110 primarily affects DNA conformation. The binding of purified MelR protein to a melAB promoter fragment carrying both binding sites has also been studied: binding results in four retarded bands in gel assays. Methylation interference experiments have been exploited to identify the binding sites occupied in each complex. Although both binding sites share a common 18 bp sequence, MelR binding to the more upstream site is stronger. We could find no evidence for co-operative interactions between MelR and RNA polymerase and no major effects of melibiose. Some evidence for melibiose-dependent distortion in complexes between MelR and the melAB promoter is discussed.
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PMID:Studies on the binding of the Escherichia coli MelR transcription activator protein to operator sequences at the MelAB promoter. 144 8

The opening of duplex DNA in the elongation phase of transcription by Escherichia coli RNA polymerase in vivo was detected at a regulatory site where a prolonged pause in transcription occurs. Single-stranded DNA in the transcription bubble was identified by its reactivity with potassium permanganate (KMnO4). The elongation structure in vivo was similar to that of transcription complexes made in vitro with some differences. The observed reactivity to KMnO4 of the DNA template strand was consistent with the existence of an RNA-DNA hybrid of about 12 nucleotides.
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PMID:Structure of transcription elongation complexes in vivo. 153 8

A region upstream from the Escherichia coli rrnB P1 promoter, the upstream activator region (UAR), increases the activity of the promoter in vivo and the rate of association with RNA polymerase (E sigma 70) in vitro in the presence of the two initiating nucleotides. We have used four types of chemical and enzymatic footprinting probes to determine whether rrnB P1-E sigma 70 complexes formed in the presence of the initiating nucleotides (RPinit) differ from typical open complexes (RPo) formed in the absence of the initiating nucleotides and to examine the structural differences between rrnB P1 complexes containing the UAR and those lacking the UAR. We find that the rrnB P1-RPinit complex closely resembles open complexes formed at other E sigma 70 promoters, indicating that the formation of the first phosphodiester bond does not result in a major rearrangement of the promoter-RNA polymerase complex. An unusual potassium permanganate modification at position -18 in both RPo and RPinit indicates the possible presence of a subtle difference in the -10, -35 spacer structure compared to some other E. coli promoters. We show that the E sigma 70-rrnB P1 complex formed with the promoter containing the UAR has DNase I and hydroxyl radical cleavage patterns in the -50 region different from those observed with the same promoter lacking the UAR. These results are interpreted to indicate that E sigma 70 may interact with a region further upstream from that contacted by RNA polymerase bound at most other promoters and/or that unusual structural properties of this region are induced by bound E sigma 70.
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PMID:Factor-independent activation of Escherichia coli rRNA transcription. II. characterization of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase. 165 94


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