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)

The high affinity branched-chain amino acid transport system (LIV-I) in Pseudomonas aeruginosa is composed of five components: BraC, a periplasmic binding protein for branched-chain amino acids; BraD and BraE, integral membrane proteins; BraF and BraG, putative nucleotide-binding proteins. By using a T7 RNA polymerase/promoter system we overproduced the BraD, BraE, BraF, and BraG proteins in Escherichia coli. The proteins were found to form a complex in the E. coli membrane and solubilized from the membrane with octyl glucoside. The LIV-I transport system was reconstituted into proteoliposomes from solubilized proteins by a detergent dilution procedure. In this reconstituted system, leucine transport was completely dependent on the presence of all five Bra components and on ATP loaded internally to the proteoliposomes. Alanine and threonine in addition to branched-chain amino acids were transported by the proteoliposomes, reflecting the substrate specificity of the BraC protein. GTP replaced ATP well as an energy source, and CTP and UTP also replaced ATP partially. Consumption of loaded ATP and concomitant production of orthophosphate were observed only when BraC and leucine, a substrate for LIV-I, were added together to the proteoliposomes, indicating that the LIV-I transport system has an ATPase activity coupled to translocation of branched-chain amino acids across the membrane.
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PMID:Solubilization and reconstitution of the Pseudomonas aeruginosa high affinity branched-chain amino acid transport system. 140 Apr 43

T3 and T7 phages package homologous DNA more efficiently than heterologous DNA and recombinant plasmids carrying DNA sequences necessary for DNA packaging (pac sequence). The pac sequence contains a promoter for phage RNA polymerase and transcription from the promoter is necessary for DNA packaging. T3 and T7 RNA polymerases are stringently specific for their own promoters. To examine the relationship between DNA packaging and transcription, we constructed a cleared in vitro system for packaging T3 or T7 DNA containing an ammonium sulfate fractionate of a high-speed supernatant of phage-infected cells. In the system, DNA packaging required GTP and was inhibited by the 3'-deoxy analog of GTP, ATP, or CTP. The DNA packaging activity paralleled the transcriptional activity, assayed by incorporation of [32P]UTP into acid-insoluble material. In the system, homologous DNA was packaged more efficiently than heterologous DNA, but heterologous DNA was packaged as efficiently as homologous DNA by the addition of heterologous phage RNA polymerase, demonstrating that the transcriptional specificity determines the DNA packaging specificity of T3 and T7.
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PMID:Transcription dependence of DNA packaging of bacteriophages T3 and T7. 141 5

Yeast RNA polymerase II initiation factor b copurifies with three polypeptides of 85, 73, and 50 kilodaltons and with a protein kinase that phosphorylates the carboxyl-terminal repeat domain (CTD) of the largest polymerase subunit. The gene that encodes the 73-kilodalton polypeptide, designated TFB1, was cloned and found to be essential for cell growth. The deduced protein sequence exhibits no similarity to those of protein kinases. However, the sequence is similar to that of the 62-kilodalton subunit of the HeLa transcription factor BFT2, suggesting that this factor is the human counterpart of yeast factor b. Immunoprecipitation experiments using antibodies to the TFB1 gene product demonstrate that the transcriptional and CTD kinase activities of factor b are closely associated with an oligomer of the three polypeptides. Photoaffinity labeling with 3'-O-(4-benzoyl)benzoyl-ATP (adenosine triphosphate) identified an ATP-binding site in the 85-kilodalton polypeptide, suggesting that the 85-kilodalton subunit contains the catalytic domain of the kinase.
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PMID:Cloning of a subunit of yeast RNA polymerase II transcription factor b and CTD kinase. 144

1. A protein kinase type II was purified from calf thymus chromatin using ammonium sulphate fractionation, ion exchange chromatography on DEAE and phosphocellulose and affinity chromatography on phosvitin- and casein-sepharose columns. 2. The enzyme moves as a single band in non-denaturing gel electrophoresis at pH 8.3, which coincides with the enzyme activity assayed on gel slices. 3. Sodium dodecyl sulphate gel electrophoresis shows three separate polypeptide chains having M(r) of 40,000, 38,000 and 25,000, respectively. The native M(r) was about 130,000, as measured by HPLC on Superose 12 column, suggesting a subunit structure of alpha, alpha', beta 2 type. The enzyme incubated with [gamma 32P]ATP or [gamma 32P]GTP as phosphoryl donors undergoes autophosphorylation in the M(r) = 25,000 subunit. 4. The enzyme phosphorylates casein (Km = 7 microM) and phosvitin (Km = 5 microM) but not histones and was strongly deactivated by Zn2+ ions (I50 = 0.05 mM) and heparin (I50 = 0.1 micrograms/ml). 5. The enzyme seems to be the major phosphorylating system present in the 0.35 M NaCl chromatin extract of calf thymus. The RNA polymerase II from calf thymus and RNA polymerase from E. coli are both phosphorylated by protein kinase NII. The effect of phosphorylation, which causes a remarkable increase of DNA transcription rate, was studied in vitro and extensively discussed.
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PMID:Protein kinase NII from calf thymus chromatin. Isolation, characterization and some functional properties. 145 14

The assembly of activated RNA polymerase II (pol II) transcription complexes has been investigated by assaying whether pre-assembly of intermediate complexes reduces the extended time required for start-site melting. The results show that a closed complex requiring factors IIA, IID, and the acidic activator GAL4-AH forms in a rate-limiting step. This directs the templates into a productive assembly pathway. Factor TFIIB is then added rapidly, affording further protection against diversion into nonproductive pathways. These events are followed by a series of rapid steps in which the remaining general factors are assembled onto the template, which is then melted using the energy of ATP hydrolysis.
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PMID:The acidic activator GAL4-AH can stimulate polymerase II transcription by promoting assembly of a closed complex requiring TFIID and TFIIA. 151 30

We have cloned a gene from a Salmonella typhimurium with the ability to complement the rfaC mutation (heptose-deficient lipopolysaccharide, sensitivity to rough-specific bacteriophages, and susceptibility to hydrophobic antibiotics). A 1018-base pair EcoRV-Tth111I fragment, subcloned into the pBluescriptKS+ vector to yield pKZ103, retains complementing activity. Nucleotide sequencing revealed an open reading frame corresponding to a protein of 317 amino acids (M(r) approximately 35,100). The plasmid pKZ103, which has a properly aligned T7 promoter, can overexpress a protein of M(r) = 31,000 when T7 RNA polymerase is supplied. An in vitro system was established for analysis of heptose addition to the precursor [4'-32P](KDO)2-IVA (Brozek, K. A., Hosaka, K., Robertson, A. D., and Raetz, C. R. H. (1989) J. Biol. Chem, 264, 6956-6966). Soluble fractions from wild-type or heptose-deficient rfa mutants were tested for their ability to convert [4'-32P](KDO)2-IVA to more polar substances. In wild-type extracts, these conversions required addition of ATP or ADP-heptose. In extracts of rfaC-, rfaD-, or rfaE-deficient strains, no polar products were observed with ATP. ADP-heptose restored synthesis in rfaD and rfaE but not rfaC extracts, indicating that rfaD and rfaE are involved in ADP-heptose formation. When the cloned rfaC gene was introduced into an rfaC-deficient mutant, extracts from such cells regained the ability to metabolize [4'-32P](KDO)2-IVA, showing that rfaC encodes the enzyme that attaches the proximal heptose to lipopolysaccharide.
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PMID:The rfaC gene of Salmonella typhimurium. Cloning, sequencing, and enzymatic function in heptose transfer to lipopolysaccharide. 152 14

The prokaryotic activator protein NTRC binds to enhancer-like elements and activates transcription in response to nitrogen limitation by catalysing open complex formation by sigma 54 RNA polymerase holoenzyme. Formation of open complexes requires the phosphorylated form of NTRC and the reaction is ATP dependent. We find that NTRC has an ATPase activity which is activated by phosphorylation and is strongly stimulated by the presence of DNA containing specific NTRC binding sites.
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PMID:The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent. 153 52

A gene of Bacillus brevis HPD31 analogous to the Escherichia coli lon gene has been cloned and characterized. The cloned gene (B. brevis lon gene) encodes a polypeptide of 779 amino acids with a molecular weight of 87,400 which resembles E. coli protease La, the lon gene product. Fifty-two percent of the amino acid residues of the two polypeptides were identical. The ATP-binding sequences found in E. coli protease La were highly conserved. The promoter of the B. brevis lon gene resembled that recognized by the major RNA polymerase of Bacillus subtilis and did not contain sequences homologous to the E. coli heat shock promoters. The B. brevis lon gene was inactivated by insertion of the neomycin resistance gene. A mutant B. brevis carrying the inactivated lon gene showed diminished ability for the degradation of abnormal polypeptides synthesized in the presence of puromycin.
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PMID:Cloning, characterization, and inactivation of the Bacillus brevis lon gene. 155 46

We investigated the accuracy of the insertion process in RNA chain elongation catalyzed by wheat germ RNA polymerase II. Error frequencies varied from 1 misinserted nucleotide per 250 polymerized correct substrates to less than 1 in 2 x 10(5), depending on template sequence and nature of the divalent metal cofactor. Higher error ratios were observed in the presence of Mn2+ compared to Mg2+, and with alternating poly[d(G-C)].poly[d(G-C)] compared to poly[d(A-T)].poly[d(A-T)]. In this latter case the eukaryotic RNA polymerase was as accurate as Escherichia coli RNA polymerase holo-enzyme. The fidelity of wheat germ RNA polymerase II was also examined in transcription of polynucleotide templates in the poly[d(G-C)] family adopting either the right-handed B or left-handed Z conformations. Error ratios for noncomplementary ATP increased markedly under experimental conditions favoring the B-to-Z conformational transition of the alternating copolymers. In accordance with the results of previous studies, the rate of productive elongation, i.e. the synthesis of poly[r(G-C)], was depressed, suggesting that the decreased accuracy of the enzyme derived from an altered competence of the enzyme to form elongation complexes on the left-handed DNA. As judged by the large difference in apparent Km values of the enzyme for complementary and noncomplementary nucleoside triphosphates, part of the discrimination between substrates seemed to take place at the initial binding step. Furthermore, the results indicate that wheat germ RNA polymerase II was able to elongate a primer with a 3'-terminal mismatch, and thus to incorporate the mismatched nucleotide stably in the nascent RAN. However, the probability of productive RNA chain elongation was much lower with noncognate than with the complementary substrates.
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PMID:Accuracy of wheat-germ RNA polymerase II. General enzymatic properties and effect of template conformational transition from right-handed B-DNA to left-handed Z-DNA. 158 82

The positive control protein NifA of Klebsiella pneumoniae activates transcription by RNA polymerase containing sigma 54 by catalysing open promoter complex formation. We show that the integrity of the putative ATP-binding pocket in the central domain of NifA is necessary for the positive control function of NifA, but is not required for DNA-binding or recognition of NifA by NifL. The inactive mutant NifA proteins are trans dominant to wild-type NifA and are unable to catalyse formation of open promoter complexes irrespective of whether a closed promoter complex at the nifH promoter has preformed. Formation of the closed complex results in a DNA structural distortion adjacent to the DNA region melted in the open promoter complex. This distortion lies at the leading edge of the E sigma 54 footprint. Although unable to catalyse open complex formation, some mutant NifAs altered the chemical reactivity of the distorted base-pair indicating that they retain the ability to recognize the closed promoter complex. The activation phenotype of partially active NifA molecules was sensitive to promoter sequences known to influence closed complex formation, indicating differences in (1) the susceptibility of the closed complexes towards activation and (2) their requirements for NifA during activation.
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PMID:Central domain of the positive control protein NifA and its role in transcriptional activation. 159 20


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