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 factor is a heat-stable protein found in large amounts in Escherichia coli. In vitro, this protein has been found to stimulate transcription of lambda templates by E. coli DNA-dependent RNA polymerase. The subunit molecular weight of this factor has been re-estimated and found to be 15500 +/- 1000 in the presence of 2-mercaptoethanol. Crosslinking experiments performed with dimethylsuberimidate in the presence or absence of the DNA template indicate the presence of multiples of the 15500-Mr subunit up to the tetramer, the dimeric species being predominant. One cysteinyl residue per 15000-Mr subunit is labeled by 4-chloro-7-nitrobenzofurazan. This residue is not labeled if the factor is exposed to oxidizing conditions. In this case, three lysyl residues are titrated. H1 factor behaves as a DNA-binding protein. We have detected binding to DNA by two independent methods: displacement of a fluorescently labeled factor by the native protein and retention of radioactive DNA on millipore filters in the presence of the factor. Under our experimental conditions (high ionic strength, absence of magnesium ions), the saturation function of lambda plac DNA as well as of wild-type lambda DNA has been found to be non-cooperative. Saturation is reached when 300 +/- 30 molecules of dimeric factor are bound per lambda molecule, the average dissociation constant of the complex being 10nM. The dissociation time of the H1.DNA complex is less than 5 s at 37 degrees C. The binding of this factor lowers the affinity of native DNA for ethidium bromide. In the presence of this intercalating dye, the solubility of the complex decreases drastically.
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PMID:Physico-chemical properties of a DNA binding protein: Escherichia coli factor H1. 33 3

1. The perturbing effect of glycerol on the direct spectrum of Escherichia coli DNA-dependent RNA polymerase has been studied. 2. By comparison with model compounds and with the unfolded polymerase in 3.8m-urea it was possible to determine the ratio of tyrosine and tryptophan residues present. On reduction of the urea-treated enzyme with 2-mercaptoethanol, no further change in the difference spectrum occurred. 3. The amino acid composition of the enzyme is given. 4. In the intact protein approx. 30% of the tryptophan and 54% of the tyrosine residues were exposed. In conjunction with the extinction value and molecular weight this corresponded to 7 tryptophan residues and 57 tyrosine residues on the surface and 16 tryptophan residues and 48 tyrosine residues ;buried'. 5. The optical rotatory dispersion of the enzyme was unaffected by 20% glycerol. 6. The helix content calculated from Moffit plots over 560-300nm was 13%, and from the 233nm trough 13%.
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PMID:Location of aromatic amino acids and belix content in Escherichia coli ribonucleic acid polymerase. 494 33

The effects of 14 metal ions (chlorides) on the transcription of calf thymus DNA and phage T4 DNA with Escherichia coli RNA polymerase were tested. These assays were conducted under improved conditions of lower pH and in the absence of 2-mercaptoethanol to permit greater stability of the metal ions in solution. Among the divalent metal ions tested, the concentration-dependent order of inhibition of overall transcription is Pb2+ greater than Zn2+ greater than Cu2+ greater than Be2+ greater than Cd2+ greater than Ni2+ greater than Ca2+ greater than Co2+ greater than Mn2+ greater than Mg2+ greater than Sr2+ and is the same with either template. At pH 7.4 and in the absence of 2-mercaptoethanol, considerably lower concentrations of several of the divalent metal ions are needed for inhibition of overall transcription than at pH 8.1 and in the presence of 2-mercaptoethanol. Ca2+, Mg2+, Sr2+, Zn2+, Li+, Na+, and K+--considered to be non-mutagenic and non-carcinogenic--decrease chain initiation (measured with T4 DNA) at concentrations that inhibit overall transcription. Pb2+, Cd2+, Co2+, Be2+, and Mn2+--all mutagenic or carcinogenic--stimulate chain initiation (although at widely different rates) at concentrations that inhibit overall transcription. Cu2+ and Ni2+--both carcinogenic--stimulate initiation only at very low concentrations, followed by a progressive decrease in initiation at concentrations that inhibit overall transcription.
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PMID:Selective effects of metal ions on RNA synthesis rates. 617 51

Hantaan virus, the prototype virus of hemorrhagic fever with renal syndrome, was examined for nucleic acid characteristics which would support its previously proposed inclusion in the virus family Bunyaviridae. Nucleocapsid RNA from Hantaan virions and a control bunyavirus were examined for ribonuclease A (RNase A) sensitivity. Both viruses exhibited a similar accessibility of RNA within nucleocapsids to digestion by RNase A. Complete digestion of the RNA of both viruses was affected with high concentrations of ribonuclease. Evidence for negative strand RNA polarity was obtained by an in vitro transcriptase assay. RNA dependent RNA polymerase activity was associated with Hantaan virions. Polymerase activity required manganese and nucleoside triphosphates and was enhanced by magnesium, 2-mercaptoethanol, and sodium chloride. Oligonucleotide map analysis of the large (L), medium (M), and small (S) genome segments of Hantaan virus demonstrated that each RNA species was unique with respect to each other and was different from host cell ribosomal RNA. A common 3' terminal sequence of the three genome segments was determined to be 3' AUCAUCAUCUG. This sequence is different from those reported for viruses within the four recognized genera of the Bunyaviridae. Because all other data were consistent with nucleic acid characteristics of the Bunyaviridae, we propose a separate genus within the Bunyaviridae with Hantaan as its prototype virs.
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PMID:Analysis of Hantaan virus RNA: evidence for a new genus of bunyaviridae. 641 60

Crude microsomes from lactating rabbit mammary gland were incubated with prolactin. The incubation mixture was centrifuged and the supernatant was incubated with isolated mammary cell nuclei from lactating rabbits treated for 4 days by bromocryptin to antagonize prolactin and to deinduce casein gene transcription. Nuclei were incubated with HgCTP, and the newly synthesized mercurated RNA was isolated on SH-Sepharose columns. The content of beta-casein mRNA sequences in the fraction eluted with 2-mercaptoethanol was estimated with a [(3)H]cDNA probe obtained from partially purified beta-casein mRNA. The supernatant markedly stimulated beta-casein gene transcription but not 28S rRNA transcription. The same effect was obtained with other lactogenic hormones such as human growth hormone and ovine placental lactogen but was not observed with bovine growth hormone, insulin, parathyroid hormone, luteotropic hormone, or epidermal growth factor. Prolactin and human growth hormone were totally inactive when added directly to nuclei. The factor stimulating beta-casein gene transcription was also generated by membranes containing prolactin receptors such as those from liver, ovary, adrenals, and brain but not by membranes from heart, lung, and muscle, which do not bind prolactin. The factor stimulated beta-casein transcription when added to mammary nuclei from pseudopregnant or bromocryptin-treated lactating rabbits, in which the transcription rate is submaximal, but was ineffective on mammary nuclei prepared from untreated fully lactating rabbits. The factor was unable to induce beta-casein gene transcription in nuclei isolated from rabbit liver and reticulocytes. The factor did not stimulate the transcription of globin genes in nuclei isolated from reticulocytes or the transcription of mammary "housekeeping" genes evaluated by a cDNA probe prepared from total mRNA isolated from an unstimulated mammary gland. The transcription of beta-casein genes was abolished by adding alpha-amanitin to the medium in the presence or in the absence of the factor, indicating that the generation of mercurated beta-casein mRNA sequences depended upon the transcriptional activity of RNA polymerase II. The addition of the factor to the incubation mixture did not enhance total and alpha-amanitin-sensitive RNA synthesis. These data suggest that the binding of prolactin to its receptor in vitro induces the formation of a second messager, which specifically stimulates the transcription of prolactin-sensitive genes.
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PMID:Prolactin induces release of a factor from membranes capable of stimulating beta-casein gene transcription in isolated mammary cell nuclei. 388 13

The effect of disulfide and sulfhydryl reagents on the rate of abortive and productive elongation has been studied using Escherichia coli RNA polymerase holoenzyme and poly[d(A-T)] as template. In the presence of UTP as a single substrate and UpA as a primer, the enzyme catalyzed efficiently the synthesis of the trinucleotide product UpApU. Incubation of RNA polymerase with 1 mM 2-mercaptoethanol resulted in a 5-fold increase of the rate of UpApU synthesis. In contrast, incubation of the enzyme with 1 mM 5,5'-dithio-bis(2-nitrobenzoic) acid resulted in a 6-fold decrease of the rate of abortive elongation. Determination of the steady state kinetic constants associated with UpApU synthesis disclosed that the disulfide and sulfhydryl reagents mainly affected the rate of UpApU release from the ternary transcription complexes and therefore influenced the stability of such complexes.
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PMID:Effect of disulfide and sulfhydryl reagents on abortive and productive elongation catalyzed by Escherichia coli RNA polymerase. 773 58

In an effort to better understand protein-protein photoaffinity cross-linking using aryl azides, we have tested a number of factors influencing the cross-linking of the sigma 70 subunit of Escherichia coli RNA polymerase to core RNA polymerase. These factors include the effect of the incubations necessary for the derivatization of the protein on enzyme activity, the effect of overhead lighting on azide stability, the effect of reducing agents on azide stability, aggregation of the derivatized protein, and a comparison of two types of aryl azide cross-linkers, N-[(5-azido-2-nitrobenzoyl)oxy]succinimide (ANB-NOS) and (N-hydroxysuccinimidyl)-4-azidosalicylic acid (NHS-ASA). We found that derivatization proceeds effectively in a buffer similar to the buffer used during protein purification, that overderivatization can cause protein aggregation, that room lighting does not appreciably destroy aryl azides, and that 0.1 mM DTT is a better choice of reducing agent than 5 mM 2-mercaptoethanol. The cross-link products were separated by SDS gel electrophoresis and identified on Western blots by cross-reactivity with monoclonal antibodies to the individual subunits of RNA polymerase. In agreement with previous work (Coggins et al., 1977; Hillel & Wu, 1977), it was possible to cross-link sigma 70 to all three of the subunits of RNA polymerase. With a combination of gel analysis, chemical cleavage, and immunodetection, it is possible to demonstrate that sigma 70 cross-links to the alpha subunit between residues 209 and 329.
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PMID:Use of aryl azide cross-linkers to investigate protein-protein interactions: an optimization of important conditions as applied to Escherichia coli RNA polymerase and localization of a sigma 70-alpha cross-link to the C-terminal region of alpha. 791 30

From sequence comparisons between the tobramovirus genomes an open reading frame (ORF-X) potentially encoding a small, positively charged protein (33- to 45-amino-acids long) was found to overlap the immediate 3' and 5' sides of the transport protein gene and coat protein gene, respectively. In vitro translation of the monocistronic artificial transcripts generated with T7 RNA polymerase yielded a protein of M(r) 4000 (p4) and an unexpected trypsin-sensitive complex of M(r) 54,000 that was resistant to reduction with 2-mercaptoethanol but could be dissociated by 8 M urea. Assembly of this complex was inhibited completely by site-directed mutagenesis within a conserved, positively charged 5-amino-acid long segment of the ORF-X protein. After centrifugation in low salt buffer the 54-kDa complex remained mostly associated with ribosomes. Apparently this complex represents a specific aggregate of the p4 product of ORF-X with a protein of approximate M(r) 50,000 that is a component of the translation apparatus.
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PMID:A novel open reading frame in tobacco mosaic virus genome coding for a putative small, positively charged protein. 828 38