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 relationship between NS protein phosphorylation and RNA polymerase activities was determined in nucleocapsids purified from vesicular stomatitis virus grown in BHK cells. Phosphate incorporation into endogenous NS protein under transcription conditions reached a maximum value of 0.06 mol/mol of NS within 20 to 30 min, while RNA synthesis remained linear for 90 min. Phosphate incorporation into NS increased further upon addition of kinase-free NS protein but not upon addition of nucleocapsid kinase (prepared as described below), indicating that cessation of NS phosphorylation under transcribing conditions was due to substrate exhaustion. When NS was phosphorylated with 32P, less than 8% of the radiolabel was lost during subsequent transcription, indicating that this phosphate did not turn over. Treatment of nucleocapsids with 5'-p-fluorosulfonylbenzoyl adenosine resulted in greater than 90% inhibition of NS phosphorylation but had no effect on RNA polymerase activity. Fast protein liquid (Superose-6) chromatography of a nucleocapsid (L + NS) fraction resulted in complete separation of the viral (L + NS) protein from NS-phosphorylating activity. The addition of this kinase-free (L + NS) fraction to a kinase-deficient N-RNA fraction reconstituted an active RNA polymerase containing less than 20% of the original NS-phosphorylating activity. These results demonstrate that NS-phosphorylating activity is unnecessary during vesicular stomatitis virus RNA synthesis and indicate that all of the protein kinase(s) present in purified nucleocapsids is probably of cellular rather than viral origin.
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PMID:Phosphorylation of NS protein by vesicular stomatitis virus nucleocapsids: lack of effect during RNA synthesis and separation of kinase from L protein. 216 40

Evidence is presented that isoproterenol treatment of rat C6 glioma cells, under conditions that increase glioma cell cAMP levels, causes the phosphorylative modification of several RNA polymerase II subunits. RNA polymerase II in control and isoproterenol-stimulated 32Pi-labeled confluent glioma cells was immunoprecipitated from ribonuclease-treated nuclear extracts with hen anti-calf RNA polymerase II antiserum conjugated to Sepharose. The immunoprecipitated RNA polymerase II was analyzed for 32P-labeled subunits by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Using this technique, we have shown that isoproterenol causes a time-dependent increase of phosphate incorporation into RNA polymerase II subunits of 214,000, 180,000, 140,000, 35,000, 28,000, and 16,500 daltons. Phosphate incorporation occurred exclusively on serine in all of the six subunits. About 0.5-2 mol of phosphate/mol of RNA polymerase II subunit were incorporated. Dibutyryl cAMP (10(-3)M) mimics the stimulatory action of isoproterenol and mediates increased phosphate incorporation into the six subunits. (RS)-propranolol (10(-4)M) prevents the isoproterenol-mediated phosphorylative changes. These data indicate that isoproterenol, via cAMP, mediates a transient structural modification of RNA polymerase II subunits in rat C6 glioma cells which may possibly lead to a modulation of RNA polymerase II function(s).
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PMID:Phosphorylation of rat C6 glioma cell DNA-dependent RNA polymerase II in vivo. Identification of phosphorylated subunits and modulation of phosphorylation by isoproterenol and N6,O2'-dibutyryl cyclic AMP. 609 70

We demonstrate that it is possible to simultaneously resolve both an mRNA and its protein product by electrophoresis in a single SDS-polyacrylamide gel by using double labeling with [32P]H3PO4 and [35S]methionine, and an elongated 5% stacking gel atop the 10% resolving gel. The mRNA is resolved in the 5% gel; the protein, as expected, resolves in the 10% gel. Using a T7 expression system, we show that putative mRNA bands in the 5% gel are: 1) labeled only with 32P and not with 35S; 2) inducible with isopropylthiogalactopyranoside (needed to induce a T7 RNA polymerase gene under control of a lac promoter); 3) synthesized in the presence of rifampicin (T7 RNA polymerase is not inhibited by rifampicin); 4) degraded by base or RNase treatment; and 5) are largely resistant to DNase treatment. The mRNA bands were also evident in samples not treated with rifampicin. We used this technique to confirm previously published results that inhibition of expression by consecutive low-usage AGG arginine codons inserted near the 5' end of a test message in Escherichia coli is at the level of translation.
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PMID:Use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis to resolve mRNA and its protein product in one gel. 936 50

Contacts of phosphate groups at positions -12, -15, and -18 in relation to the transcription initiation site in the non-template strand of lac UV5 promoter with lysines or histidines of E. coli RNA polymerase in the open complex model were studied. A number of synthetic oligonucleotides from the -10-area of the non-template strand containing activated 5'-terminal phosphate group were cross-linked with holo- or core-enzyme of RNA polymerase. 5'-N-Hydroxybenzotriazole phosphodiesters of oligonucleotides were used as phosphate activated derivatives. They are capable of phosphorylating amino groups of lysines and histidines in the enzyme molecule that are brought into proximity with activated phosphate in the complex, resulting in the formation of a covalent bond between the oligonucleotide and the protein. The analysis of the products of cross-linking allowed the protein subunit and the amino acid residue taking part in the formation of the covalent bond for each oligonucleotide to be identified. It was found that all oligonucleotides from the non-template strand of promoter in the complex with the holo-enzyme are bound with the sigma70-subunit. When analyzing the products of partial cleavage of the complexes cross-linked at cysteines and methionines using SDS-PAGE, it was shown that phosphate at position -12 made contacts with His180 or His242 of the sigma70-subunit, the reactive amino acid residue being located between the first and second conservative regions. Phosphate at position -15 is located near lysines from two different areas--between Met413 and Met456 (regions 2.3 and 2.4) and between Met470 and Met507 (region 3.1). Phosphate at position -18 makes preferential contacts with a lysine situated between Met470 and Met507 (region 3.1). Based on the analysis of contacts of phosphate groups and the structure of the isolated sigma70-subunit established previously, a scheme of the mutual arrangement of the oligonucleotide and the sigma70-subunit possessed by the holo-enzyme has been proposed.
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PMID:Probing contacts of phosphate groups of oligonucleotides from the non-template strand of lac UV5 promoter with E. coli RNA polymerase using regioselective cross-linking. 1088 81

The Chlamydomonas reinhardtii plastid and mitochondrial transcriptomes were surveyed for changes in RNA profiles resulting from growth in 12 culture conditions representing 8 abiotic stimuli. Organellar RNA abundance exhibited marked changes during nutrient stress and exposure to UV light, as revealed by both RNA gel blot and DNA microarray analyses. Of particular note were large increases in tufA and clpP transcript abundance during nutrient limitation. Phosphate and sulfur limitation resulted in the most global, yet opposite, effects on organellar RNA abundance, changes that were dissected further using run-on transcription assays. Removal of sulfate from the culture medium, which is known to reduce photosynthesis, resulted in 2-fold to 10-fold decreases in transcription rates, which were reflected in lower RNA abundance. The decrease in transcriptional activity was completely reversible and recovered to twice the control level after sulfate replenishment. Conversely, phosphate limitation resulted in a twofold to threefold increase in RNA abundance that was found to be a post-transcriptional effect, because it could be accounted for by increased RNA stability. This finding is consistent with the known metabolic slowdown under phosphate stress. Additionally, inhibitor studies suggested that unlike those in higher plants, Chlamydomonas chloroplasts lack a nucleus-encoded plastid RNA polymerase. The apparently single type of polymerase could contribute to the rapid and genome-wide transcriptional responses observed within the chloroplast.
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PMID:The Chlamydomonas reinhardtii organellar genomes respond transcriptionally and post-transcriptionally to abiotic stimuli. 1595 33

In this study, the phosphoproteome of Corynebacterium glutamicum, an industrially important soil bacterium of the Corynebacterium/Mycobacterium/Nocardia (CMN) group of Gram-positive bacteria, was investigated by two different detection methods: first, by in vivo radio-labeling using [(33)P]-phosphoric acid with subsequent autoradiography and second, by immunostaining with phosphoamino acid-specific monoclonal antibodies. After two-dimensional gel electrophoresis (2-DE), around 60 [(33)P]-labeled protein spots were visualized and around 90 antibody-decorated protein spots detected; 31 of the protein spots were detected with both methods. By peptide mass fingerprinting, 41 different proteins were identified, namely 5-enolpyruvylshikimate 3-phosphate synthase, aconitase, acyl-CoA carboxylase, acyl-CoA synthetase, ATP (synthase alpha- and beta-chain), carbamoyl-phosphate synthase, citrate synthase, cysteine synthase, DnaK, the elongation factors G, P, Ts and Tu, enolase, fructose bisphosphate aldolase, fumarase, Gap dehydrogenase, glutamine synthetase I, glycine hydroxymethyltransferase, GroEL2, GTPase, heat-inducible transcriptional repressor DnaJ2, inorganic pyrophosphatase, isocitrate dehydrogenase, ketol-acid reductoisomerase, lactate dehydrogenase, leucine-tRNA ligase, lipoamide dehydrogenase, methionine synthase, O-acetylhomoserine sulfhydrylase, pyruvate carboxylase, pyruvate kinase, pyruvate oxidase, ribosomal protein S1, RNA polymerase (beta-subunit), succinyl-CoA:CoA transferase, transketolase and UDP-N-acetylmuramoyl-L-alanine ligase, besides a hypothetical 35k protein and a hypothetical glucose kinase. Both detection techniques were used to create a phosphoproteome map. Additionally, the influence of nitrogen deprivation on the phosphoproteome of C. glutamicum was investigated.
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PMID:Towards a phosphoproteome map of Corynebacterium glutamicum. 1292 88

Examination of mutant and knockout phenotypes with altered phosphate/pyrophosphate distribution has demonstrated that cementum, the mineralized tissue that sheathes the tooth root, is very sensitive to local levels of phosphate and pyrophosphate. The aim of this study was to examine the potential regulation of cementoblast cell behavior by inorganic phosphate (P(i)). Immortalized murine cementoblasts were treated with P(i) in vitro, and effects on gene expression (by quantitative real-time reverse-transcriptase polymerase chain reaction [RT-PCR]) and cell proliferation (by hemacytometer count) were observed. Dose-response (0.1-10 mM) and time-course (1-48 hours) assays were performed, as well as studies including the Na-P(i) uptake inhibitor phosphonoformic acid. Real-time RT-PCR indicated regulation by phosphate of several genes associated with differentiation/mineralization. A dose of 5 mM P(i) upregulated genes including the SIBLING family genes osteopontin (Opn, >300% of control) and dentin matrix protein-1 (Dmp-1, >3,000% of control). Another SIBLING family member, bone sialoprotein (Bsp), was downregulated, as were osteocalcin (Ocn) and type I collagen (Col1). Time-course experiments indicated that these genes responded within 6-24 hours. Time-course experiments also indicated rapid regulation (by 6 hours) of genes concerned with phosphate/pyrophosphate homeostasis, including the mouse progressive ankylosis gene (Ank), plasma cell membrane glycoprotein-1 (Pc-1), tissue nonspecific alkaline phosphatase (Tnap), and the Pit1 Na-P(i) cotransporter. Phosphate effects on cementoblasts were further shown to be uptake-dependent and proliferation-independent. These data suggest regulation by phosphate of multiple genes in cementoblasts in vitro. During formation, phosphate and pyrophosphate may be important regulators of cementoblast functions including maturation and regulation of matrix mineralization.
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PMID:Regulation of cementoblast gene expression by inorganic phosphate in vitro. 1646 74

In vitro nuclear protein phosphorylation is enhanced in nuclei isolated from 2,4-dichlorophenoxyacetic acid (2,4-d)-treated mature soybean (Glycine max) hypocotyl relative to nuclei from untreated tissue. Increased nuclear protein phosphorylation correlates with increased levels of nuclear protein kinase activity. These changes generally parallel previously reported 2,4-d-enhanced RNA polymerase activity of these nuclei and the in vivo levels of RNA synthesis. Phosphate incorporation represents bona fide protein phosphorylation, with 87% of the label being identified as phosphoserine and 7% as phosphothreonine. Label from [gamma-(32)P]adenosine 5'-triphosphate is incorporated primarily into various nonhistone fractions with the greatest accumulation in loosely associated fractions (either released during incubation with ATP or removed by 0.15 m Nacl). Although electrophoretic analysis on sodium dodecyl sulfate gels shows no differences in the protein profiles of the loosely associated or sodium dodecyl sulfate-soluble nonhistone proteins, there are changes in the pattern of phosphorylation of other proteins, after 2,4-d treatment. Acid-soluble basic nuclear proteins are phosphorylated to a much lower extent than are the other nuclear protein fractions. While histone F(1) is subject to slight phosphorylation when nuclei are labeled in vitro, phosphorylation of the other histones is undetectable. One acid-soluble protein shows a substantial increase in quantity and in phosphorylation after 2,4-d treatment. This protein is similar in electrophoretic mobility to pea histone F(1) but its identity is unknown. Urea-acetic acid gels of the acid-soluble nuclear proteins show that auxin treatment results in increased quantities and in increased phosphorylation of various low mobility nonhistone basic nuclear proteins.
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PMID:2,4-Dichlorophenoxyacetic Acid-enhanced Phosphorylation of Soybean Nuclear Proteins. 1666 Feb 58

Failures in fracture healing are mainly caused by a lack of vascularization. Adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, have been reported to differentiate into osteoblasts in vitro; however, the therapeutic potential of CD34+ cells for fracture healing is still unclear. Therefore, we performed a series of experiments to test our hypothesis that functional fracture healing is supported by vasculogenesis and osteogenesis via regenerative plasticity of CD34+ cells. Peripheral blood CD34+ cells, isolated from total mononuclear cells of adult human volunteers, showed gene expression of osteocalcin in 4 of 20 freshly isolated cells by single cell reverse transcriptase-polymerase chain reaction analysis. Phosphate-buffered saline, mononuclear cells, or CD34+ cells were intravenously transplanted after producing nonhealing femoral fractures in nude rats. Reverse transcriptase-polymerase chain reaction and immunohistochemical staining at the peri-fracture site demonstrated molecular and histological expression of human-specific markers for endothelial cells and osteoblasts at week 2. Functional bone healing assessed by biomechanical as well as radiological and histological examinations was significantly enhanced by CD34+ cell transplantation compared with the other groups. Our data suggest circulating human CD34+ cells have therapeutic potential to promote an environment conducive to neovascularization and osteogenesis in damaged skeletal tissue, allowing the complete healing of fractures.
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PMID:Therapeutic potential of vasculogenesis and osteogenesis promoted by peripheral blood CD34-positive cells for functional bone healing. 1700 98

Advances in structure determination have made possible the analysis of large macromolecular complexes (some with nearly 10,000 residues, such as GroEL). The large-scale conformational changes associated with these complexes require new approaches. Historically, a crucial component of motion analysis has been the identification of moving rigid blocks from the comparison of different conformations. However, existing tools do not allow consistent block identification in very large structures. Here, we describe a novel method, RigidFinder, for such identification of rigid blocks from different conformations-across many scales, from large complexes to small loops. RigidFinder defines rigidity in terms of blocks, where inter-residue distances are conserved across conformations. Distance conservation, unlike the averaged values (e.g., RMSD) used by many other methods, allows for sensitive identification of motions. A further distinguishing feature of our method, is that, it is capable of finding blocks made from nonconsecutive fragments of multiple polypeptide chains. In our implementation, we utilize an efficient quasi-dynamic programming search algorithm that allows for real-time application to very large structures. RigidFinder can be used at a dedicated web server (http://rigidfinder.molmovdb.org). The server also provides links to examples at various scales such as loop closure, domain motions, partial refolding, and subunit shifts. Moreover, here we describe the detailed application of RigidFinder to four large structures: Pyruvate Phosphate Dikinase, T7 RNA polymerase, RNA polymerase II, and GroEL. The results of the method are in excellent agreement with the expert-described rigid blocks.
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PMID:RigidFinder: a fast and sensitive method to detect rigid blocks in large macromolecular complexes. 1970 87


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