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)

New improved methods were developed for the purification to apparent homogeneity of alpha, beta, beta', and sigma subunits of Escherichia coli RNA polymerase (RNAP) from corresponding overproducing strains. The purified subunits were assembled into enzymatically active RNAP holoenzyme (alpha 2 beta beta' sigma) using the optimal subunit molar ratio (alpha:beta:beta':sigma = 2:8:4:1) at a total protein concentration of 0.5 mg/ml. The presence of sigma subunit and 10 microM ZnCl2 in the reconstitution mixture increased the yield of RNAP approximately 4 times. The assembled RNA polymerase was purified by two successive chromatographic steps using size-exclusion Superose 6 and anion exchange Mono Q FPLC columns, which resulted in the electrophoretically homogeneous holoenzyme with overall yield of 56%. The specific activity of the recombinant RNAP estimated by the standard T4 transcription assay was 6.5 nmol of [3H]UTP incorporated into acid-insoluble RNA product per microgram of RNAP per 1 h.
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PMID:Recombinant Escherichia coli RNA polymerase: purification of individually overexpressed subunits and in vitro assembly. 828 46

Escherichia coli RNA polymerase is composed of four different subunits, 2 alpha, beta, beta' and sigma. Among these subunits, the role of beta' is poorly understood. The rpoC10 mutation affecting beta' has been isolated as a suppressor mutation of the temperature-sensitive nusA11 mutant. DNA sequence analysis revealed that the rpoC10 mutant is a substitution of Lys for Glu-402. This increased positive charge appears to compensate for the increased negative charge present in the nusA11 protein (Asp for Gly-181). In vivo measurements of reporter gene expression have revealed that rpoC10 restores rho-dependent termination but fails to restore rho-independent termination in nusA11. Moreover, the rpoC10 mutation, in combination with any nusA mutation, inhibited lambda Q-mediated antitermination without affecting N antitermination and severely restricted lambda phage development. The inhibition of Q function and lambda growth could be compensated for by overproducing Q. These results suggest that the RNA polymerase beta' subunit plays a crucial role in factor-dependent transcription termination and antitermination.
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PMID:Pleiotropic effects of the rpoC10 mutation affecting the RNA polymerase beta' subunit of Escherichia coli on factor-dependent transcription termination and antitermination. 841 81

In the present study, we analyzed human adult brain, fetal spinal cord, and an interleukin-2 (IL-2)-responsive human oligodendroglioma subclone, TC620.6A2, for the presence of mRNAs for the alpha, beta, and gamma chains of the interleukin-2 receptor (IL-2R alpha, IL-2R beta, and IL-2R gamma). IL-2R beta mRNA, but not IL-2R alpha or IL-2R gamma was detectable by Northern blot analysis in adult human brain tissues. Northern blot analysis of TC620.6A2 and human fetal tissues revealed mRNAs of 1.5 kb and 1.3 kb that hybridized to the IL-2R alpha cDNA at low to medium stringency. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments were done on the TC620.6A2 cell line utilizing primers to IL-2R alpha, IL-2R beta, and IL-2R gamma. Southern blot analysis of the TC620.6A2 RT-PCR reactions detected products identical in size to the peripheral blood lymphocyte (PBL) positive controls at high stringency. Several of the TC620.6A2 IL-2R alpha, IL-2R beta, and IL-2R gamma cDNAs were cloned and sequenced. The sequences were found to be identical to the known IL-2R sequences. To our knowledge, these experiments are the first to demonstrate the presence of authentic IL-2R mRNAs in a human oligodendrocyte-like cell line. Demonstration of mRNA for IL-2R beta in human adult brain, IL-2R alpha in fetal brain, and IL-2R alpha, IL-2R beta, and IL-2R gamma in a malignant neural cell line suggests the possibility of a role for IL-2/IL-2R interactions in development and disease.
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PMID:Molecular cloning of IL-2R alpha, IL-2R beta, and IL-2R gamma cDNAs from a human oligodendroglioma cell line: presence of IL-2R mRNAs in the human central nervous system. 853 Jan 86

We have cloned the gene for a putative chloroplast RNA polymerase sigma factor from the unicellular rhodophyte Cyanidium caldarium. This gene contains an open reading frame encoding a protein of 609 amino acids with domains highly homologous to all four conserved regions found in bacterial and cyanobacterial sigma 70-type subunits. When Southern blots of genomic DNA were hybridized to the "rpoD box" oligonucleotide probe, up to six hybridizing hands were observed. Transcripts of the sigma factor gene were undetectable in RNA from dark-grown cells but were abundant in the poly(A)+ fraction of RNA from illuminated cells. The sigma factor gene was expressed in Escherichia coli, and antibodies against the expressed sigma factor fusion protein cross-reacted with a 55-kDa protein in partially purified chloroplast RNA polymerase. Antibodies directed against a cyanobacterial RNA polymerase sigma factor also cross-reacted with a 55-kDa protein in the same enzyme preparation. Immunoprecipitation experiments showed that this enzyme preparation contains proteins with the same molecular weights as the alpha, beta, beta', and beta" subunits of chloroplast RNA polymerase in higher plants. This study identifies a gene for a plastid RNA polymerase sigma factor and indicates that there may be a family of nuclear-encoded sigma factors that recognize promoters in subsets of plastid genes and regulate differential gene expression at the transcriptional level.
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PMID:Molecular characterization of a positively photoregulated nuclear gene for a chloroplast RNA polymerase sigma factor in Cyanidium caldarium. 862 35

Numerous physical and genetic approaches have identified residues in the alpha, beta, beta' and sigma subunits of Escherichia coli RNA polymerase that are involved in transcriptional processes; in contrast, relatively little data exist to demonstrate interacting regions within or between the subunits themselves. As a means of identifying regions in the beta subunit that may interact, we have sought intragenic suppressor mutations of a class of elongation-defective and termination-proficient inviable rpoB alleles that affect highly conserved residues. We obtained intragenic allele-specific suppressors of GD566 (located in conserved region D) and AV676 (located in conserved region E). With one exception, these allele-specific suppressors also map to highly conserved regions of the beta subunit. Allele specific suppression is a genetic criterion for protein-protein interaction. Moreover, the functional properties of the mutants suggests that suppression is likely to result from protein-protein interaction rather than from functional compensation. Our suppression studies provide evidence for the interaction of conserved regions B and D as well as conserved regions E and H of the beta polypeptide. We suggest that these, as well as other conserved regions of the beta polypeptide, may interact with each other to provide a framework for the function of the enzyme.
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PMID:Identifying interacting regions in the beta subunit of Escherichia coli RNA polymerase. 862 20

Control of RNA polymerase is a common means of regulating gene expression. A detailed picture of both the structure and how the structural details of RNA polymerase encode function is a key to understanding the molecular strategies used to regulate RNA polymerase. We review here data which ascribes functions to some regions of the primary sequence of the subunits (alpha, beta beta' sigma) which make up E. coli RNA polymerase. We review both genetic and biochemical data which place regions of the primary sequence that are distant from one another in close proximity in the tertiary structure. Finally we discuss the implications of these findings on the quaternary structure of RNA polymerase.
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PMID:A structure/function analysis of Escherichia coli RNA polymerase. 873 69

Microtubules play an essential role in cell division. Little is known about possible variations of total tubulin and tubulin isotype expression during the cell cycle. We analyzed the total tubulin content, tubulin polymerization status and tubulin isotype content in resting and dividing human K562 leukemic cells and human MES-SA sarcoma cells. Although the total cellular tubulin content increases as the cells progress toward mitosis, the total tubulin/total protein ratio is stable during the cell cycle. Reverse transcriptase-polymerase chain reaction was applied to analyze the levels of expression of alpha, beta, and gamma-tubulin isotypes. Whereas alpha-tubulin isotype and gamma-tubulin transcripts were found to be expressed at constant levels throughout the cell cycle, some of the beta-tubulin isotype transcripts were found to be more highly expressed in dividing then in resting cells. Both of the class IV beta-tubulin isotype transcripts (human 5 beta and beta 2, Class IVa and IVb, respectively) were expressed in dividing K562 and MES-SA cells at twice the levels found in resting cells. Increased expression of the class IV isotype proteins in dividing cells was confirmed by immunoblotting, both in K562 and in MES-SA cells. A larger fraction of total cell tubulin was found to be polymerized in dividing cells (36-40%) than in resting cells (27-30%). The degree of polymerization of class IV tubulin in dividing and resting cells was similar to that of total tubulin. These results show that total tubulin is expressed as constant levels throughout the cell cycle but that the degree of polymerization is increased as cells are committed to division. The relative overexpression of the two class IV beta-tubulin isotypes in dividing cells suggests functional specificity for these isotypes and a regulatory role of these isotypes on the microtubule network during mitosis.
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PMID:Differential expression of tubulin isotypes during the cell cycle. 887 65

The DNA-dependent RNA polymerases from heat-shocked and vegetatively grown cells of Bacillus subtilis were isolated and compared. The RNA polymerase from non-stressed cells had the well known alpha, beta, beta' and sigma composition of eubacterial RNA polymerases. The RNA polymerase from heat-shocked cells exhibited one additional band shown by SDS-PAGE. N-terminal sequencing of the first 16 amino acids of the associated protein demonstrated its identity with the ribosomal protein S2.
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PMID:Copurification of ribosomal protein S2 and DNA-dependent RNA polymerase from heat-shocked cells of Bacillus subtilis. 909 Jan 22

Pulmonary epithelial Na+ channels (ENaC), composed of three distinct subunits (alpha, beta, and gamma), play a critical role in the regulation of fluid reabsorption from airspaces of late-gestation fetal lung. We studied the expression of ENaC subunit genes in cultured human fetal lung. All three mRNAs were expressed at low levels in second trimester lung (13-32% of adult values at 24 wk gestation). There was a spontaneous increase of approximately threefold over preculture values of all three subunits within 24 h of explant culture in serum-free Waymouth's medium. Dexamethasone (Dex) induced all three mRNAs by two- to threefold. Maximal induction was noted by 8 h with 30-100 nM Dex and half-maximal stimulation with 3-10 nM Dex. Cycloheximide decreased basal expression of all three subunits by 8 h but did not alter the response to Dex. Actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), inhibitors of RNA polymerase II, decreased the basal and the Dex-induced expression of all three subunits with a more marked effect on human hENaC-gamma than on hENaC-alpha or hENaC-beta. Under conditions where transcription was blocked by actinomycin D or DRB, Dex did not alter the stability of the three mRNAs. Triiodothyronine (T3) at low (2 nM) or high (100 nM) concentrations had no effect on the expression of the three subunits in the presence or absence of low (10 nM) or high (100 nM) concentrations of Dex for 8 or 24 h. Similarly, 8-bromoadenosine 3',5'-cyclic monophosphate (2 microM) had no effect on basal or Dex-induced increase in the three subunits. We conclude that the three Na+ channel subunit genes are expressed in second trimester human fetal lung and are coordinately upregulated by glucocorticoid hormones but not by T3 or adenosine 3',5'-cyclic monophosphate. Glucocorticoid induction is receptor mediated, is primarily transcriptional, and does not require the induction of an intermediate protein for transcriptional enhancement. We speculate that induction of lung ENaC may contribute to the beneficial effects of antenatal glucocorticoids in premature babies.
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PMID:Glucocorticoid regulation of epithelial sodium channel genes in human fetal lung. 925 60

The Escherichia coli genome encodes genes for seven different sigma subunit species while only having single genes for the alpha, beta, and beta' subunits that make up the RNA polymerase core enzyme. The various sigma factors compete for binding to the core enzyme, upon which they confer promoter DNA-specific transcription initiation to the polymerase. We have mapped a major interaction site between one of the sigma species, sigma70, and beta'. Using far-Western blotting analysis of chemically cleaved and genetically engineered protein fragments, we have identified a N-terminal fragment of beta' (residues 60-309) that could bind sigma70. We were able to more precisely map the interaction domain to amino acid residues 260-309 of beta' using nickel nitrilotriacetic acid co-immobilization assays.
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PMID:Localization of a sigma70 binding site on the N terminus of the Escherichia coli RNA polymerase beta' subunit. 981 48


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