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)

H1 protein, a heat-stable low-molecular-weight DNA-binding factor previously described by Cukier-Kahn et al. [Proc. Nat. Acad. Sci USA (1972) 69, 3643-3647] markedly stimulates in vitro synthesis of lac-specific RNA directed by bacteriophage lambdah80 dlac or phi80 dlac DNA templates in the presence of purified E. coli RNA polymerase holoenzyme. The extent of stimulation obtained by addition of H1 alone is usually greater than that observed with the cAMP receptor protein-cAMP combination. H1 effect varies quite appreciably (from 4- to 16-fold) with the functional state of the promoter, being much larger with lambdah80 dlac p-s, a transducing DNA carrying a superpromoter mutation, than with lambdah80 dlac p+. H1 and cAMP receptor protein effects are nearly additive, although interpretation of the data obtained at high H1 concentration is complicated by the appearance of some inhibitory property. While the cAMP-receptor-protein-mediated synthesis is asymmetrical ("I" strand almost exclusively copied), the degree of asymmetry observed with H1 is less pronounced, suggesting asymmetrical copying from the lac promoter and symmetric transcription from other regions of the DNA. Synthesis of lac-specific RNA from lambdah80 dtrp/lac or phi80 dlac p-r uv5 templates, in which lac promoters are insensitive to cAMP receptor protein, either as a result of lac fusion to the trp operon or mutation in the lac promoter, is totally H1-insensitive. Glycerol (10-15% w/w) can fully substitute for H1 in stimulating lac RNA synthesis in a fashion analogous to that reported for the cAMP receptor protein-cAMP system. The possibility that H1 acts by causing conformational modifications at the promoter level in a way that increases its functional state, and that this effect is more pronounced with operons sensitive to cAMP receptor protein, is discussed.
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PMID:Effect of a low-molecular-weight DNA binding protein, H1 factor, on the in vitro transcription of the lactose operon in Escherichia coli. 16 21

Class III DNA-dependent RNA polymerase (EC 2.7.7.6) was highly purified from cauliflower (Brassica oleracea, var. bortytis) by using polyethyleneimine precipitation. The specific activity of the enzyme was comparable to that reported for mammalian enzymes. Glycerol gradient sedimentation analysis indicated that the sedimantation coefficient (23 S) was slightly higher than that of enzyme II from cauliflower. The class III enzyme was inhibited by alpha-amanitin at high concentrations (50% inhibition at 200 microgram/ml). The Km value for nucleoside triphosphate was determined. Template specificities for single synthetic polymers showed that the enzyme read pyrimidine homopolymers as templates and preferred poly(dT) to poly(dC). The enzyme transcribed both strands of homopolymer pairs of poly(dI). poly(dC) and poly(dA).poly(dT). The synthetic polyribonucleotides were not effectively read. Competition experiments with these synthetic polymers indicated that the enzyme had different binding specificities which were not the same as their template specificities. The different binding affinities and template specificites for synthetic templates of the three classes of enzyme suggest that the enzyme can discriminate among different template sequences.
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PMID:DNA-dependent RNA polymerase III from cauliflower. Characterization and template specificity. 62 58

sigma 70, encoded by rpoD, is the major sigma factor in Escherichia coli. rpoD285 (rpoD800) is a small deletion mutation in rpoD that confers a temperature-sensitive growth phenotype because the mutant sigma 70 is rapidly degraded at high temperature. Extragenic mutations which reduce the rate of degradation of RpoD285 sigma 70 permit growth at high temperature. One class of such suppressors is located in rpoH, the gene encoding sigma 32, an alternative sigma factor required for transcription of the heat shock genes. One of these, rpoH113, is incompatible with rpoD+. We determined the mechanism of incompatibility. Although RpoH113 sigma 32 continues to be made when wild-type sigma 70 is present, cells show reduced ability to express heat shock genes and to transcribe from heat shock promoters. Glycerol gradient fractionation of sigma 32 into the holoenzyme and free sigma suggests that RpoH113 sigma 32 has a lower binding affinity for core RNA polymerase than does wild-type sigma 32. The presence of wild-type sigma 70 exacerbates this defect. We suggest that the reduced ability of RpoH113 sigma 32 to compete with wild-type sigma 70 for core RNA polymerase explains the incompatibility between rpoH113 and rpoD+. The rpoH113 cells would have reduced amounts of sigma 32 holoenzyme and thus be unable to express sufficient amounts of the essential heat shock proteins to maintain viability.
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PMID:A mutant sigma 32 with a small deletion in conserved region 3 of sigma has reduced affinity for core RNA polymerase. 162 56

Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II.
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PMID:Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly. 171 23

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19 + 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of 3H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115-120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19 + 2.1. Primers synthesized using M13mp19 + 2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.
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PMID:Pea chloroplast DNA primase: characterization and role in initiation of replication. 186 57

The steroid-binding domain of the human glucocorticoid receptor was expressed in Escherichia coli either as a fusion protein with protein A or under control of the T7 RNA polymerase promoter. The recombinant proteins were found to bind steroids with the normal specificity for a glucocorticoid receptor but with reduced affinity (Kd for triamcinolone acetonide approximately 70 nM). Glycerol gradient analysis of the E. coli lystate containing the recombinant protein indicated no interaction between the glucocorticoid receptor fragment and heat shock proteins. However, synthesis of the corresponding fragments of glucocorticoid receptor in vitro using rabbit reticulocyte lystate resulted in the formation of proteins that bound triamcinolone acetonide with high affinity (Kd 2nM). Glycerol gradient analysis of these proteins, with and without molybdate, indicated that the in vitro synthesised receptor fragments formed complexes with hsp90 as previously shown for the full-length rat glucocorticoid receptor. Radiosequence analysis of the recombinant steroid-binding domain expressed in E. coli and affinity labelled with dexamethasone mesylate identified binding of the steroid to Cys-638 predominantly. However, all cysteine residues within the steroid-binding domain were affinity labelled to a certain degree indicating that the recombinant protein has a structure similar to the native receptor but more open and accessible.
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PMID:The steroid-binding properties of recombinant glucocorticoid receptor: a putative role for heat shock protein hsp90. 227 31

Using isolated nuclei prepared from influenza virus-infected HeLa cells, factors affecting the synthesis of two species of positive-sense RNA transcripts, i.e., mRNA and cRNA (complementary RNA to vRNA) were analyzed. In the presence of low concentrations of salt, both mRNA and cRNA were synthesized, whereas in the presence of high concentrations of salt, mRNA was synthesized predominantly. Salt-extracts of nuclei (NE) mainly produced cRNA while mRNA was a major product synthesized by salt-treated nuclei (delta N). In the presence of high concentrations of salt, the NE produced mRNA instead of cRNA. After centrifugation of the NE, the precipitates (NEP) predominantly produced mRNA while the supernatant (NES) alone exhibited a low level of cRNA synthesis activity. With the addition of the NES fraction, mRNA synthesis by the NEP was switched to cRNA synthesis. Glycerol gradient centrifugation of the NES fraction in the presence of high salt yielded vRNA-RNA polymerase complexes that catalyzed mRNA synthesis. These observations indicate that a regulatory factor(s) that can be dissociated from vRNA-RNA polymerase complexes upon exposure to high ionic strength is involved in the switch from mRNA to cRNA synthesis. This activity was not detected in nuclear extracts prepared from uninfected cells, suggesting that such a factor(s) is either encoded by the virus genome or induced by virus infection.
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PMID:In vitro synthesis of influenza viral RNA: biochemical complementation assay of factors required for influenza virus replication. 280 17

Two general transcription factors (IIE and IIB) (TF) were purified from HeLa cell nuclear extracts and shown to be absolutely required, along with two additional factors (IIA and IID) and RNA polymerase II, for specific transcription initiation at the adenovirus major late promoter. TFIIB and TFIIE were also required, in addition to TFIIA, TFIID, RNA polymerase II, and the adenovirus 2 major late promoter, for the formation of a (preinitiation) complex that could initiate transcription (upon addition of nucleoside triphosphates) in the presence of heparin concentrations which inhibited the action of unbound factors. Glycerol gradient analyses indicated independent interactions of TFIIE with TFIIB and with the purified RNA polymerase II, but not with RNA polymerase III. Transcription factors IIB and IIE were also shown to be required for specific initiation of transcription from several cellular and viral class II promoters.
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PMID:Factors involved in specific transcription by mammalian RNA polymerase II. Purification and functional analysis of initiation factors IIB and IIE. 302 9

A DNA-dependent RNA polymerase that transcribes vaccinia virus early genes was partially purified from virus cores by deoxycholate extraction and DEAE-cellulose column chromatography. Accurately initiated and terminated RNAs were synthesized by this enzyme in the presence of a linear duplex DNA template. Glycerol gradient sedimentation resolved the in vitro transcription system into two components: fraction I, a rapidly sedimenting RNA polymerase that initiated transcription at an early promoter but transcribed beyond the in vivo 3' terminus to yield a run-off transcript, and fraction II, a more slowly sedimenting fraction, itself devoid of RNA polymerase, that restored efficient termination when added back to fraction I. The termination factor was heat-labile, resistant to N-ethylmaleimide, and did not exhibit endonucleolytic activity on run-off transcripts. Factor-dependent termination required specific sequence information upstream of the site of termination. The vaccinia termination factor was purified extensively by column chromatography on DEAE-cellulose, heparin-agarose, phosphocellulose, and DNA-agarose, and by velocity sedimentation in a glycerol gradient. At each step, termination factor copurified with the vaccinia mRNA capping enzyme. The preparation was well over 90% pure with respect to the latter enzyme, suggesting that termination activity was tightly associated with, if not intrinsic to, the capping enzyme. Nonetheless, formation of the 5'-cap structure did not appear to be a prerequisite for termination.
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PMID:Purification and characterization of a transcription termination factor from vaccinia virions. 362 64

Captan (N-trichloromethylthiocyclohex-4-ene-1,2-dicarboximide) was shown to inhibit RNA synthesis in vitro catalysed by Escherichia coli RNA polymerase. Incorporation of [gamma-32P]ATP and [gamma-32P]GTP was inhibited by captan to the same extent as overall RNA synthesis. The ratio of [3H]UTP incorporation to that of [gamma-32P]ATP or of [gamma-32P]GTP in control and captan-treated samples indicated that initiation was inhibited, but the length of RNA chains being synthesized was not altered by captan treatment. Limited-substrate assays in which re-initiation of RNA chains did not occur also showed that captan had no effect on the elongation reaction. Studies which measured the interaction of RNA polymerase with template DNA revealed that the binding of enzyme to DNA was inhibited by captan. Glycerol-gradient sedimentation of the captan-treated RNA polymerase indicated that the inhibition of the enzyme was irreversible and did not result in dissociation of its subunits. These data are consistent with a mechanism in which RNA polymerase activity was irreversibly altered by captan, resulting in an inability of the enzyme to bind to the template. This interaction was probably at the DNA-binding site on the polymerase and did not involve reaction of captan with the DNA template.
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PMID:Mechanism of inhibition of Escherichia coli RNA polymerase by captan. 617 15


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