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)

Better understanding of hemostasis will be possible by the identification of new lineage-specific stimuli that regulate platelet formation. We describe a novel functional megakaryocyte receptor that belongs to a family of ionotropic glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype responsible for synaptic neurotransmission in the central nervous system (CNS). Northern blotting and reverse-transcriptase polymerase chain reaction (RT-PCR) studies identified expression of NMDAR1 and NMDAR2D type subunit mRNA in rat marrow, human megakaryocytes, and MEG-01 clonal megakaryoblastic cells. Immunohistochemistry and in vivo autoradiographic binding of the NMDA receptor-specific antagonist MK-801 confirmed that megakaryocytes expressed open channel-forming NMDA receptors in vivo. Western blots indicated that megakaryocyte NMDAR1 was either unglycosylated or only glycosylated to low levels, and of identical size to CNS-type NMDAR1 after deglycosylation with endoglycosidase F/peptide-N-glycosidase F. In functional studies, we demonstrated that NMDA receptor activity was necessary for phorbol myristate acetate (PMA)-induced differentiation of megakaryoblastic cells; NMDA receptor blockade by specific antagonists significantly inhibited PMA-mediated increases in cell size, CD41 expression, and adhesion of MEG-01 cells. These results provide evidence for a novel pathway by which megakaryocytopoiesis and platelet production may be regulated.
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PMID:Expression of a functional N-methyl-D-aspartate-type glutamate receptor by bone marrow megakaryocytes. 1021 82

Cell-free transcription of archaeal promoters is mediated by two archaeal transcription factors, aTBP and TFB, which are orthologues of the eukaryotic transcription factors TBP and TFIIB. Using the cell-free transcription system described for the hyperthermophilic Archaeon Pyrococcus furiosus by Hethke et al., the temperature limits and template topology requirements of archaeal transcription were investigated. aTBP activity was not affected after incubation for 1 hr at 100 degrees. In contrast, the half-life of RNA polymerase activity was 23 min and that of TFB activity was 3 min. The half-life of a 328-nt RNA product was 10 min at 100 degrees. Best stability of RNA was observed at pH 6, at 400 mm K-glutamate in the absence of Mg(2+) ions. Physiological concentrations of K-glutamate were found to stabilize protein components in addition, indicating that salt is an important extrinsic factor contributing to thermostability. Both RNA and proteins were stabilized by the osmolyte betaine at a concentration of 1 m. The highest activity for RNA synthesis at 95 degrees was obtained in the presence of 1 m betaine and 400 mm K-glutamate. Positively supercoiled DNA, which was found to exist in Pyrococcus cells, can be transcribed in vitro both at 70 degrees and 90 degrees. However, negatively supercoiled DNA was the preferred template at all temperatures tested. Analyses of transcripts from plasmid topoisomers harboring the glutamate dehydrogenase promoter and of transcription reactions conducted in the presence of reverse gyrase indicate that positive supercoiling of DNA inhibits transcription from this promoter.
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PMID:Cell-free transcription at 95 degrees: thermostability of transcriptional components and DNA topology requirements of Pyrococcus transcription. 1043 May 63

Mutation of glutamate 62 to lysine in yeast transcription factor (TF) IIB (Sua7) causes a cold-sensitive phenotype. This mutant also leads to preferential transcription of downstream start sites on some promoters in vivo. To explore the molecular nature of these phenotypes, the TFIIB E62K mutant was characterized in vitro. The mutant interacts with TATA-binding protein normally. In three different assays, the mutant can also interact with RNA polymerase II and recruit it and the other basal transcription factors to a promoter. Despite the ability to assemble a transcription complex, the TFIIB E62K protein is severely defective in transcription in vitro. Therefore, the role of TFIIB must be more than simply bridging TATA-binding protein and polymerase at the promoter. We propose that the region around Glu-62 in yeast TFIIB plays a role in start site selection, perhaps mediating a conformational change in the polymerase or the DNA during the search for initiation sites. This step may be related to the yeast-specific spacing between TATA elements and start sites since mutations of the corresponding glutamate in mammalian TFIIB do not produce a similar effect.
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PMID:Evidence that transcription factor IIB is required for a post-assembly step in transcription initiation. 1046 20

The promoter selectivity of two extracytoplasmic function (ECF) subfamily sigma subunits, sigma(E) (sigma(24)) and sigma(FecI) (sigma(18)), of Escherichia coli RNA polymerase was analyzed by using an in vitro transcription system and various promoters. The Esigma(E) holoenzyme recognized only the known cognate promoters, rpoEP2, rpoHP3, and degP, and the Esigma(FecI) recognized only one known cognate promoter, fecA. The strict promoter recognition properties of sigma(E) and sigma(FecI) are similar to those of other minor sigma subunits. Transcription by Esigma(E) and Esigma(FecI) was enhanced by high concentrations of glutamate, as in the case of other minor sigma subunits. The optimum temperature for transcription by Esigma(FecI) was low, around 25 degrees C, apparently in agreement with the high rate of iron sequestration by E. coli at low temperatures. By quantitative Western blot analysis, the intracellular levels of sigma(E) and sigma(FecI) in the uninduced steady-state culture of E. coli W3110 (type A) were determined to be 0.7 to 2.0 and 0.1 to 0.2 fmol per microg of total proteins (or 3 to 9 and 0.4 to 0.9 molecules per cell), respectively, and less than 1% of the level of the major sigma(70) subunit.
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PMID:Two extracytoplasmic function sigma subunits, sigma(E) and sigma(FecI), of Escherichia coli: promoter selectivity and intracellular levels. 1064 50

The ability of metabotropic glutamate receptor activation to mobilise intracellular calcium was investigated in cultured dorsal root ganglion (DRG) neurones from neonatal rats using the calcium sensitive fluorescent dye Fura-2. L-glutamate (10 microM) caused sustained and oscillatory increases in intracellular calcium concentration ([Ca2+]i) in a subpopulation of cultured DRG neurones. The oscillatory responses were not blocked by combined application of the ionotropic glutamate receptor antagonists MK 801 (2 microM) and CNQX (20 microM). Oscillations in [Ca2+]i were also observed following application of the nonselective metabotropic glutamate receptor (mGluR) agonist, trans-(1S,3R)-1-aminocyclopentane-1S, 3R-dicarboxylic acid (1S,3R)-ACPD, 20 microM) and the mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 500 microM). These responses were blocked by the selective Group I mGluR antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) (100 microM) and Ca2+ release channel inhibitors ryanodine (100 microM) and dantrolene (10 microM). The predominantly Group II agonist (2S,2'R,3'R)-2-(2'3'-dicarboxy-cyclopropyl)glycine (DCG-IV, 100 microM) failed to produce Ca2+ transients alone but suppressed responses to CHPG. Reverse transcriptase PCR techniques, using primers specific to Group I mGluRs, revealed the presence of mGluR5 but not mGluR1 mRNA in these cells. Therefore, glutamate can cause a slowly activating and reversible mobilisation of [Ca2+]i in sensory neurones by activation of ionotropic receptors, and can induce oscillatory calcium transients by selectively activating metabotropic glutamate receptors that are likely to be of the mGluR5 subtype.
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PMID:Mobilisation of intracellular Ca2+ by mGluR5 metabotropic glutamate receptor activation in neonatal rat cultured dorsal root ganglia neurones. 1072 83

sigma(38) (or sigma(S), the rpoS gene product) is a sigma subunit of RNA polymerase in Escherichia coli and directs transcription from a number of stationary-phase promoters as well as osmotically inducible promoters. In this study, we analyzed the function of the carboxy-terminal 16-amino-acid region of sigma(38) (residues 315 to 330), which is well conserved among the rpoS gene products of enteric bacterial species. Truncation of this region was shown to result in the loss of sigma activity in vivo using promoter-lacZ fusion constructs, but the mutant sigma(38) retained the binding activity in vivo to the core enzyme. The in vitro transcription analysis revealed that the transcription activity of sigma(38) holoenzyme under high potassium glutamate concentrations was significantly decreased by the truncation of the carboxy-terminal tail element.
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PMID:A carboxy-terminal 16-amino-acid region of sigma(38) of Escherichia coli is important for transcription under high-salt conditions and sigma activities in vivo. 1091 98

BC1 RNA is a neuronal cell-specific RNA polymerase III (Pol III) transcript. The BC1 RNA gene has plural types of Pol III promoters, in addition to which an E-box sequence (E2 site) acts as a transcriptional activator, which is recognized by a brain-specific protein(s). Using an in vitro transcription system, we found that the upstream region of the BC1 RNA gene contained a sequence that interfered with the activity of the E-box element in a distance-independent manner. A tandem repeat within this sequence, which was weakly homologous with the neuron-restrictive silencer element (NRSE) found in the Pol II system, was recognized by a brain nuclear protein. Consistently, the transcriptional activity increased by deleting the tandem repeat sequence. We called this BC1 RNA-repressing element BCRE. The DNA-binding specificities of BCRE-binding protein differed from that of NRSE-binding protein (NRSF). A similar protein with an ability to bind to BCRE was also found in liver and kidney. Furthermore, the glutamate analog kainic acid increased the DNA-binding of both E2 site-binding protein and BCRE-binding protein, and then the levels of BC1 RNA also increased transiently. Our results suggested that both positive and negative regulatory elements contribute to neuronal BC1 RNA expression.
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PMID:Identification of a negative regulatory DNA element for neuronal BC1 RNA expression by RNA polymerase III. 1097 16

The biological significance of glycogen accumulation and how the process is regulated in Chlamydia trachomatis remains poorly defined. C. trachomatis-infected HeLa cells were cultured in medium containing various glucose concentrations (0, 0.1, 1 or 10 mg ml-1) or in the presence of gluconeogenic carbon sources (20 mM glutamate, 20 mM malate, 20 mM alpha-ketoglutarate or 20 mM oxaloacetate), and the effects of these different culture conditions on the production of infectious chlamydial elementary bodies and glycogen accumulation were monitored. When chlamydiae were cultured in glucose concentrations greater than 1 mg ml-1, optimal growth and maximal glycogen accumulation occurred. In contrast to uninfected HeLa cells, which increased their glycogen stores when grown in the presence of high glucose concentrations, chlamydial glycogen accumulation remained essentially constant. When cultured in medium supplemented with either reduced glucose concentrations or any of the gluconeogenic carbon sources, chlamydiae still grew; however, the yield of elementary bodies was substantially decreased, and there was no significant amount of glycogen accumulated by host HeLa cells or C. trachomatis. This suggests that glycogen accumulation may not be essential for chlamydial survival. Reverse transcriptase-polymerase chain reaction (RT-PCR) results indicated that, despite the fact that the source and amount of carbon available in the medium affected chlamydial glycogen accumulation, the expression of genes required for glycogen metabolism was not significantly changed. Similarly, the expression of several genes encoding key enzymes of central metabolism was not affected by alterations in carbon source or availability. Taken together, the data suggest that, unlike most free-living bacteria, chlamydia are unable to alter the expression of genes involved in carbon metabolism in response to changes in environmental conditions.
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PMID:Regulation of carbon metabolism in Chlamydia trachomatis. 1102 87

Long-lasting alterations of neuronal functions could involve mechanisms associated with consolidation of transient extracellular signals through modulation of de novo synthesis of particular functional proteins in the brain. In eukaryotes, protein de novo synthesis is mainly under the control at the level of gene transcription by transcription factors in the cell nucleus. Transcription factors are nuclear proteins with an ability to recognize particular core nucleotides at the upstream and/or downstream of target genes, and thereby to modulate the activity of RNA polymerase II that is responsible for the formation of mRNA from double stranded DNA. Gel retardation electrophoresis is widely employed for conventional detection of DNA binding activities of a variety of transcription factors with different protein motifs. Extracellular ionotropic glutamate (Glu) signals lead to rapid and selective potentiation of DNA binding of the nuclear transcription factor activator protein-1 (AP1) that is a homo- and heterodimeric complex between Jun and Fos family members, in addition to inducing expression of the corresponding proteins, in a manner unique to each Glu signal in murine hippocampus. Therefore, extracellular Glu signals may be differentially transduced into the nucleus to express AP1 with different assemblies between Jun and Fos family members, and thereby to modulate de novo synthesis of the individual target proteins at the level of gene transcription in the hippocampus. Such mechanisms may be operative on synaptic plasticity as well as delayed neuronal death through consolidation of alterations of a variety of cellular functions induced by transient extracellular signals in the brain.
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PMID:Consolidation of transient ionotropic glutamate signals through nuclear transcription factors in the brain. 1116 2

The sigmaS and sigma70 subunits of Escherichia coli RNA polymerase recognize very similar promoter sequences. Therefore, many promoters can be activated by both holoenzymes in vitro. The same promoters, however, often exhibit distinct sigma factor selectivity in vivo. It has been shown that high salt conditions, reduced negative supercoiling and the formation of complex nucleoprotein structures in a promoter region can contribute to or even generate sigmaS selectivity. Here, we characterize the first positively acting sigmaS-selective feature in the promoter sequence itself. Using the sigmaS-dependent csiD promoter as a model system, we demonstrate that C and T at the -13 and -14 positions, respectively, result in strongest expression. We provide allele-specific suppression data indicating that these nucleotides are contacted by K173 in region 2.5 of sigmaS. In contrast, sigma70, which features a glutamate at the corresponding position (E458), as well as the sigmaS(K173E) variant, exhibit a preference for a G(-13). C(-13) is highly conserved in sigmaS-dependent promoters, and additional data with the osmY promoter demonstrate that the K173/C(-13) interaction is of general importance. In conclusion, our data demonstrate an important role for region 2.5 in sigmaS in transcription initiation. Moreover, we propose a consensus sequence for a sigmaS-selective promoter and discuss its emergence and functional properties from an evolutionary point of view.
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PMID:What makes an Escherichia coli promoter sigma(S) dependent? Role of the -13/-14 nucleotide promoter positions and region 2.5 of sigma(S). 1125 33


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