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 subunit interactions of Escherichia coli RNA polymerase by measuring the rate of cleavage of each subunit by trypsin. We have modified a standard sodium dodecyl sulfate gel technique to include sodium tetradecyl sulfate, which results in a large separation of the beta and beta' subunits. A comparison of enzyme lacking the sigma subunit (core) with holoenzyme reveals that the presence of sigma does not alter the cleavage rates of alpha or beta, but it dramatically slows the initial cleavage of beta. Simple addition of purified sigma to core results in reconstitution of holoenzyme as measured both by glycerol gradient sedimentation and protection of beta from trypsin cleavage. However, Mg2+ is required for the beta protection but not for the binding of sigma to core. Although the enzyme reconstituted in the absence of Mg2+ and lacking the sigma-beta association is similar to holoenzyme with respect to promoter binding, it catalyzes the transition from closed to open complexes much more slowly than does enzyme reconstituted in the presence of Mg2+. Thus, the specific association between beta and sigma may be involved in the DNA melting phase of the RNA polymerase-DNA interaction. We have also examined the effects of the polyanion heparin, an RNA polymerase initiation inhibitor, on the rates of trypsin cleavage of holoenzyme subunits. Again, subunits alpha and beta' were unaffected, but heparin increased the cleavage rate of beta such that the rate was indistinguishable from the beta cleavage rate of core enzyme. Since heparin did not cause the release of sigma as measured by glycerol gradient analysis, the inhibitor may simply disrupt the sigma-beta association.
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PMID:Analysis of RNA polymerase by trypsin cleavage. Evidence for a specific association between subunits sigma and beta involved in the closed to open complex transition. 700 Jul 84

Alterations in RNA polymerase structure can be detected using initial trypsin cleavage rates as a conformational probe. Both template (poly[d(A-T) . d(A-T)] and the RNA polymerase inhibitor, heparin, alter the rates at which the subunits of the enzyme are cleaved. However, while the presence of poly[d(A-T) . d(A-T)] slows the cleavage of subunits beta, sigma, and alpha by trypsin, heparin accelerates the cleavage of beta and sigma. Furthermore, the presence of heparin does not prevent the effect of poly[d(A-T) . d(A-T)] on the beta and sigma cleavage rates. Thus, heparin does not eliminate the interaction between DNA and RNA polymerase. That heparin does alter the nature of this interaction is demonstrated by the fact that template decreases the trypsin cleavage rate of subunit alpha in the absence, but not in the presence, of heparin. Like heparin, the addition of RNA to the reaction increases the accessibility of beta and sigma to trypsin. Hence the interaction of heparin with RNA polymerase may mimic the product, rather than the template, interaction.
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PMID:Analysis of RNA polymerase by trypsin cleavage. Different structural changes produced by heparin and DNA. 703 53

SP6 is a small, virulent bacteriophage which grows on Salmonella typhimurium LT2. It is morphologically similar to Escherichia coli bacteriophage T7 and its relatives, but appears to be genetically distinct. After infection a bacteriophage-specific RNA polymerase is induced in infected cells. SP6 RNA polymerase is a stable enzyme and is easily purified to homogeneity in good overall yield. The activity resides in a single polypeptide chain of Mr = 96,000. Synthesis of RNA by SP6 RNA polymerase requires a DNA template and Mg2+ ion and is strongly stimulated by either bovine serum albumin of spermidine. Thiol-reactive reagents inhibit the enzyme, suggesting the presence of essential sulfhydryl residues. RNA synthesis requires native SP6 RNA as template; DNAs from other bacteriophages including T3 and T7 are inert; hence, SP6 RNA polymerase possesses a stringent promoter specificity similar to, but distinct from that of the other phage RNA polymerases. The SP6 RNA polymerase is also highly active in synthesis of poly(rG) with poly(dI) . (dC) as template. This reaction is unlikely to involve promoter-like sites, but it appears to reflect a general catalytic capacity of the polymerase, since cleavage of the SP6 RNA polymerase with trypsin, which completely eliminates SP6-transcribing activity, has little effect on poly(rG) synthesis. Hence, it appears that the catalytic portion of the polymerase can be separated from the RNA polymerase holoenzyme.
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PMID:Bacteriophage SP6-specific RNA polymerase. I. Isolation and characterization of the enzyme. 704 Mar 72

Chick embryos, chick embryo fibroblasts, and Rous sarcoma virus-transformed chick embryo fibroblasts contain a factor that preferentially blocks the accumulation of DNA-directed RNA polymerase II transcripts. The factor was detected by inhibition of transcription in a cell-free assay system utilizing partially purified RNA polymerase II from calf thymus, soluble factors from HeLa cells, and a purified DNA template. At low concentrations, it specifically prevents the accumulation of RNA polymerase II transcripts; at higher concentrations, it blocks the accumulation of other transcripts. The factor has been partially purified by sequential chromatography on BioRex 70, DNA-cellulose, Bio-Gel P-6, and HPX-87 from extracts of chicken embryos. The activity was resistant to treatment with trypsin, pronase, or micrococcal nuclease. A partial characterization of the molecule indicates that (i) it has an apparent molecular mass of about 200-300 daltons, (ii) it is stable at pH 2 and pH 12 and to heating at 100 degrees C, (iii) it is not extractable by ether or chloroform:methanol, (2:1, v/v), and (iv) it is labile to heating at 800 degrees C. These data suggest that it is a small, hydrolphilic compound probably organic in nature. The factor is active in a transcription assay utilizing either the Rous sarcoma virus Long Terminal Repeat promoter or the chick alpha 2 (Type I) collagen-promoter as DNA templates. The accumulation of promoter-specific transcripts is blocked in a cell-free assay utilizing either Rous sarcoma virus-chick embryo fibroblast extracts or HeLa S-100 factors and calf thymus RNA polymerase II. In the absence of S-100, the factor does not appreciably affect the accumulation of randomly initiated transcripts produced by calf thymus RNA polymerase II on a DNA template; this result indicates the factor interacts directly or indirectly with some component(s) of HeLa S-100 to prevent the accumulation of RNA.
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PMID:Chicken embryo extracts contain a factor that preferentially blocks the accumulation of RNA polymerase II transcripts in a cell-free system. 713 Jan 91

Rabbit hemorrhagic disease virus, a positive-stranded RNA virus of the family Caliciviridae, encodes a trypsin-like cysteine protease as part of a large polyprotein. Upon expression in Escherichia coli, the protease releases itself from larger precursors by proteolytic cleavages at its N and C termini. Both cleavage sites were determined by N-terminal sequence analysis of the cleavage products. Cleavage at the N terminus of the protease occurred with high efficiency at an EG dipeptide at positions 1108 and 1109. Cleavage at the C terminus of the protease occurred with low efficiency at an ET dipeptide at positions 1251 and 1252. To study the cleavage specificity of the protease, amino acid substitutions were introduced at the P2, P1, and P1' positions at the cleavage site at the N-terminal boundary of the protease. This analysis showed that the amino acid at the P1 position is the most important determinant for substrate recognition. Only glutamic acid, glutamine, and aspartic acid were tolerated at this position. At the P1' position, glycine, serine, and alanine were the preferred substrates of the protease, but a number of amino acids with larger side chains were also tolerated. Substitutions at the P2 position had only little effect on the cleavage efficiency. Cell-free expression of the C-terminal half of the ORF1 polyprotein showed that the protease catalyzes cleavage at the junction of the RNA polymerase and the capsid protein. An EG dipeptide at positions 1767 and 1768 was identified as the putative cleavage site. Our data show that rabbit hemorrhagic disease virus encodes a trypsin-like cysteine protease that is similar to 3C proteases with regard to function and specificity but is more similar to 2A proteases with regard to size.
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PMID:3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the ORF1 polyprotein and analysis of cleavage specificity. 747 37

Langerhans cells (LC) belong to the dendritic cell lineage and are the principal antigen-presenting cells of squamous epithelia. Short-term cultured LC (cLC) exhibit a marked augmented capacity to stimulate allogeneic T cells and acquire the ability to activate naive T cells, probably in relation to enhanced expression of accessory signals. In this study, we evaluated the expression of B7 costimulatory molecule (CD80) in human freshly isolated (fLC) and cLC at both the protein and mRNA level. Staining of frozen skin sections did not reveal any epidermal dendritic cell reactive with either of two different anti-B7 monoclonal antibodies. fLC in suspension did not exhibit any B7 staining as evaluated by two-color flow-cytometry analysis and immunoelectron microscopy. In contrast, LC that were cultured for 24-72 h displayed strong surface B7 reactivity with a characteristic patchy pattern. Treatment with dispase and trypsin did not reduce B7 staining of cLC. Following warming to 37 degrees C, cLC tagged with anti-B7 monoclonal antibody and gold-conjugated secondary antibody could internalize surface B7 by using the organelles of receptor-mediated endocytosis. B7 mRNA, detected by the reverse-transcriptase polymerase chain reaction technique, was expressed at a low level in purified (> 90% HLA-DR+) fLC but not in LC-depleted epidermal cells, and was markedly upregulated in purified cLC. The results indicate that 1) fLC do not express B7 protein on their surface, but acquire B7 during culture, 2) surface B7 is not sensitive to trypsin, 3) B7 expression is regulated primarily at the mRNA level, and 4) membrane B7 can be internalized within cLC. B7 molecule on CLC may be relevant to their increased antigen-presenting cell potency and ability to stimulate naive T lymphocytes.
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PMID:Expression of B7 costimulatory molecule in cultured human epidermal Langerhans cells is regulated at the mRNA level. 751 82

We have established a dermal fibroblast-like stromal cell line, DFB-1, and a clone, 12E2, from epidermal sheets prepared from the skin of BALB/c mouse ears by trypsin digestion. They were suggested to be fibroblasts or myofibroblasts, as 1) they were polygonal or spindle-shaped under the phase-contrast microscope, 2) they did not possess any tonofilaments or desmosomes, and 3) they did not express any marker for bone marrow-derived cells or macrophages. Interestingly, these cells showed a unique phenomenon of "pseudo-emperiporesis," which was first recognized in the interaction between thymic nurse cells and thymocytes. Namely, two T-cell clones and one T-cell hybridoma migrated beneath the cytoplasmic projections of the fibroblast-like cutaneous stromal cells in culture. Furthermore, secretion of interleukin 7 by these cells was confirmed by bioassay using an IL-7-dependent cell line and by inhibition with anti-interleukin 7 antibody, and the expression of interleukin 7 mRNA was also demonstrated in these cells by a combination of reverse-transcriptase polymerase chain reaction and Southern blot analysis. These data strongly suggest the presence of unique stromal cells even in the skin, probably at the upper dermis, which can function like the nurse cells in the thymus. These stromal cells may play a crucial role in cutaneous immunophysiology.
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PMID:Cultured murine dermal cells can function like thymic nurse cells. 751 55

Osmoregulated porin gene expression in Escherichia coli is controlled by the two-component regulatory system EnvZ and OmpR. EnvZ, the osmosensor, is an inner membrane protein and a histidine kinase. EnvZ phosphorylates OmpR, a cytoplasmic DNA-binding protein, on an aspartyl residue. Phospho-OmpR binds to the promoters of the porin genes to regulate the expression of ompF and ompC. We describe the use of limited proteolysis by trypsin and ion spray mass spectrometry to characterize phospho-OmpR and the conformational changes that occur upon phosphorylation. Our results are consistent with a two-domain structure for OmpR, an N-terminal phosphorylation domain joined to a C-terminal DNA-binding domain by a flexible linker region. In the presence of acetyl phosphate, OmpR is phosphorylated at only one site. Phosphorylation induces a conformational change that is transmitted to the C-terminal domain via the central linker. Previous genetic analysis identified a region in the C-terminal domain that is required for transcriptional activation. Our results indicate that this region is within a surface-exposed loop. We propose that this loop contacts the alpha subunit of RNA polymerase to activate transcription. Mass spectrometry also reveals an unusual dephosphorylated form of OmpR, the potential significance of which is discussed.
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PMID:Phosphorylation-dependent conformational changes in OmpR, an osmoregulatory DNA-binding protein of Escherichia coli. 756 33

The alpha subunit of Escherichia coli RNA polymerase plays essential roles in protein-protein contacts, not only for RNA polymerase assembly, but also for transcription activation by class I factors. To reveal the structure-function relationship of the alpha subunit, we attempted to elucidate the organization of the structural domains by analysis of the pattern of limited proteolysis with two endoproteases, V8 protease and trypsin. The results indicate that one region, Arg235 to Glu244, is highly accessible to endoproteases. We propose that the alpha subunit consists of two major structural domains, the amino-terminal domain upstream from Arg235 and the carboxy-terminal domain downstream from Glu245, each being connected by an inter-domain linker formed by the spacer between these two amino acid residues. The structural organization is in good agreement with its functional map, i.e., the amino-terminal subunit assembly determinants and the carboxy-terminal transcription activation determinants, including the contact sites with class I transcription factors and DNA UP (enhancer) elements. The secondary proteolytic cleavage sites were also determined, in order to analyse intra-domain structures.
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PMID:Structural map of the alpha subunit of Escherichia coli RNA polymerase: structural domains identified by proteolytic cleavage. 775 34

Poliovirus RNA polymerase (3Dpol) was cross-linked to [32P]ribonucleoside triphosphates (NTPs) by reduction of oxidized NTP-protein complexes. Cross-linked complexes were digested with cyanogen bromide, and resulting peptides were fractionated by reverse-phase HPLC. 32P-Labeled peptides were purified by secondary HPLC fractionation and/or additional digestion with endoproteinases Glu-C, TPCK-trypsin, or Asp-N followed by another HPLC fractionation. N-Terminal sequences of the major [32P]-peptides were determined, and approximate sizes of these peptides were obtained by SDS-polyacrylamide gel electrophoresis. Two major NTP binding sites in 3Dpol were found. One site was between Asp-266 and Met-286; possible binding residues in this fragment were Lys-276, Lys-278, or Lys-283. A second binding site was between Ala-57 and Met-74 with Lys-61 or Lys-66 as possible binding residues. Alignment of these regions on the known structure of HIV-1 reverse transcriptase allowed us to predict the position of the downstream nucleotide binding site in the conserved "fingers" subdomain present near the active site cleft of both RNA and DNA polymerases. The N-terminal nucleotide binding site is not contained within a region that is conserved among other polymerases.
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PMID:Identification of nucleotide binding sites in the poliovirus RNA polymerase. 775 55


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