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

---

Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

During infection of Escherichia coli, the phage T4 early protein AsiA inhibits open complex formation by the RNA polymerase holoenzyme Efinal sigma(70) at -10/-35 bacterial promoters through binding to region 4.2 of the final sigma(70) subunit. We used the -10/-35 lacUV5 promoter to study the properties of the Efinal sigma(70). AsiA complex in the presence of the glutamate anion. Under these experimental conditions, inhibition by AsiA was significantly decreased. KMnO(4) probing showed that the observed residual transcriptional activity was due to the slow transformation of the ternary complex Efinal sigma(70). AsiA.lacUV5 into an open complex. In agreement with this observation, affinity of the enzyme for the promoter was 10-fold lower in the ternary complex than in the binary complex Efinal sigma(70).lacUV5. A tau plot analysis of abortive transcription reactions showed that AsiA binding to Efinal sigma(70) resulted in a 120-fold decrease in the second-order on-rate constant of the reaction of Efinal sigma(70) with lacUV5 and a 55-fold decrease in the rate constant of the isomerization step leading to the open complex. This ternary complex still responded to activation by the cAMP.catabolite activator protein complex. We show that compensatory Efinal sigma(70)/promoter upstream contacts involving the C-terminal domains of alpha subunits in Efinal sigma(70) become essential for the binding of Efinal sigma(70). AsiA to the lacUV5 promoter.
...
PMID:The Escherichia coli RNA polymerase.anti-sigma 70 AsiA complex utilizes alpha-carboxyl-terminal domain upstream promoter contacts to transcribe from a -10/-35 promoter. 1127 17

Sedimentation equilibrium studies show that the Escherichia coli cyclic AMP receptor protein (CAP) and RNA polymerase holoenzyme associate to form a 2:2 complex in vitro. No complexes of lower stoichiometry (1:1, 2:1, 1:2) were detected over a wide range of CAP and RNA polymerase concentrations, suggesting that the interaction is highly cooperative. The absence of higher stoichiometry complexes, even in the limit of high [protein], suggests that the 2:2 species represents binding saturation for this system. The 2:2 pattern of complex formation is robust. A lower-limit estimate of the formation constant in our standard buffer (40 mm Tris (pH 7.9), 10 mm MgCl(2), 0.1 mm dithiothreitol, 5% glycerol, 100 mm KCl) is 2 x 10(20) m(-3). The qualitative pattern of association is unchanged over the temperature range 4 degrees C < or = T < or = 20 degrees C, by substitution of glutamate for chloride as the dominant anion, or on addition of 20 microm cAMP to the reaction mix. These results limit the possible mechanisms of CAP-polymerase association. In addition, they support the idea that CAP binding may influence the availability of the monomeric form of RNA polymerase that mediates transcription at many promoters.
...
PMID:The Escherichia coli cyclic AMP receptor protein forms a 2:2 complex with RNA polymerase holoenzyme, in vitro. 1190 95

Transient glutamate signaling often leads to long lasting and permanent alterations of a variety of cellular functions through particular membrane receptors in the brain. For elucidation of mechanisms underlying long-term consolidation of transient extracellular signals, we have examined expression and degradation of particular Fos family member proteins required for assembly to the nuclear transcription factor activator protein-1 in this study. Transcription factors could modulate the activity of RNA polymerase II responsible for the formation of mRNA from genomic DNA in the nucleus and therefore regulate de novo synthesis of particular target functional proteins. Mice were intraperitoneally injected with 100 mg/kg N-methyl-D-aspartic acid (NMDA) or 40 mg/kg kainic acid (KA), followed by homogenization of hippocampus in the presence of different protease and phosphatase inhibitors 2 h after administration, and subsequent preparation of nuclear and cytosolic fractions. The systemic administration of both NMDA and KA induced marked expression of particular Fos family members, including c-Fos and Fra-2 proteins, in hippocampal nuclear and cytosolic fractions. Incubation at 30 degrees C for 1 to 18 h led to differential degradation profiles of each Fos family member protein in nuclear fractions in a manner peculiar to the individual excitants. Degradation rate was also affected by dialysis and subsequent addition of inhibitors for phosphatases and proteases. These results suggest that in vivo NMDA and KA signals may additionally modulate the activity of heterologous machineries responsible for breakdown of each Fos family member in a unique manner in nuclear fractions, rather than cytosolic fractions, of murine hippocampus.
...
PMID:Nuclear degradation of particular Fos family members expressed following injections of NMDA and kainate in murine hippocampus. 1192 65

Omega4514 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. DNA upstream of the insertion site was cloned, and the promoter was localized. The promoter resembles vegetative promoters in sequence, and sigma(A) RNA polymerase, the major form of RNA polymerase in growing M. xanthus, initiated transcription from this promoter in vitro. Two complete open reading frames were identified downstream of the promoter and before the Omega4514 insertion. The first gene product (ORF1) has a putative helix-turn-helix DNA-binding motif and shows sequence similarity to transcriptional regulators. ORF2 is most similar to subunit A of glutaconate coenzyme A (CoA) transferase, which is involved in glutamate fermentation. Tn5 lac Omega4514 is inserted in the third codon of ORF3, which is similar to subunit B of glutaconate CoA-transferase. An orf1 disruption mutant exhibited a mild sporulation defect, whereas neither a disruption of orf2 nor insertion Omega4514 in orf3 caused a defect. Based on DNA sequence analysis, the three genes are likely to be cotranscribed with a fourth gene whose product is similar to alcohol dehydrogenases. ORF1 delays and reduces expression of the operon during development, but relief from this negative autoregulation does not fully explain the regulation of the operon, because expression from a small promoter-containing fragment is strongly induced during development of an orf1 mutant. Also, multiple upstream DNA elements are necessary for full developmental expression. These results suggest that transcriptional activation also regulates the operon. Omega4514 is the first example of a developmentally regulated M. xanthus operon that is transcribed by the major vegetative RNA polymerase, and its regulation appears to involve both negative autoregulation by ORF1 and positive regulation by one or more transcriptional activators.
...
PMID:Identification of the Omega4514 regulatory region, a developmental promoter of Myxococcus xanthus that is transcribed in vitro by the major vegetative RNA polymerase. 1202 52

Using the previously established Escherichia coli two-plasmid system, we identified a promoter recognized by the Streptomyces coelicolor stress-response sigma factor sigma(H). The promoter directed expression of the gltB gene, encoding a protein with considerable homology with large subunit of glutamate synthases. S1-nuclease mapping using RNA prepared from S. coelicolor identified an identical transcription start point corresponding to the promoter. The level of the transcript from this promoter was substantially reduced in a S. coelicolor sigH mutant. In addition to this sigH-dependent gltBp2 promoter, expression of the S. coelicolor gltB gene was directed by two other promoters, gltBp1 and gltBp3, independent upon sigH. S. coelicolor core RNA polymerase, after complementation with sigma(H), was able to recognize the gltBp2 promoter in vitro. These results suggested that the S. coelicolor gltB gene is under the control of stress-response sigma(H).
...
PMID:Stress-response sigma factor sigma(H) directs expression of the gltB gene encoding glutamate synthase in Streptomyces coelicolor A3(2). 1215 Nov 8

Amino acids at conserved sites in the residue sequence of 10 ancient proteins, from 844 phylogenetically diverse sources, were used to specify their time of origin in the interval before species divergence from the last common ancestor (LCA). The order of amino acid addition to the genetic code, based on biosynthesis path length and other molecular evidence, provided a reference for evaluating the 'code age' of each residue profile examined. Significantly earlier estimates were obtained for conserved amino acid residues in these proteins than non-conserved residues. Evidence from the primary structure of 'fossil' proteins thus corroborated the biosynthetic order of amino acid addition to the code.Low potential ferredoxin (Fdxn) had the earliest residue profile among the proteins in this study. A phylogenetic tree for 82 prokaryote Fdxn sequences was rooted midway between bacteria and archaea branches. LCA Fdxn had a 23-residue antecedent whose residue profile matched mid-expansion phase codon assignments and included an amide residue. It contained a highly acidic N-terminal region and a non-charged C-terminal region, with all four cysteine residues. This small protein apparently anchored a [4Fe-4S] cluster, ligated by C-terminal cysteines, to a positively charged mineral surface, consistent with mediating e(-) transfer in a primordial surface system before cells appeared. Its negatively charged N-terminal 'attachment site' was highly mutable during evolution of ancestral Fdxn for Bacteria and Archaea, consistent with a loss of function after cell formation. An initial glutamate to lysine substitution may link 'attachment site' removal to early post-expansion phase entry of basic amino acids to the code. As proteins evidently anchored non-charged amide residues initially, surface attachment of cofactors and other functional groups emerges as a general function of pre-cell proteins.A phylogenetic tree of 107 proteolipid (PL) helix-1 sequences from H(+)-ATPase of bacteria, archaea and eukaryotes had its root between prokaryote branches. LCA PL h1 residue profile optimally fit a late expansion phase codon array. Sequence repeats in transmembrane PL helices h1 and h2 indicated formation of the archetypal PL hairpin structure involved successive tandem duplications, initiated within the gene for an 11-residue (or 4-residue) hydrophobic peptide. Ancestral PL h1 lacked acidic residues, in a fundamental departure from the prototype pre-cell protein. By this stage, proteins with a hydrophobic domain had evolved. Its non-polar, late expansion phase residue profile point to ancestral PL being a component of an early permeable cell membrane. Other indicators of cell formation about this stage of code evolution include phospholipid biosynthesis path length, FtsZ residue profile, and late entry of basic amino acids into the genetic code. Estimates based on conserved residues in prokaryote cell septation protein, FtsZ, and proteins involved with synthesis, transcription and replication of DNA revealed FtsZ, ribonucleotide reductase, RNA polymerase core subunits and 5'-->3' flap exonuclease, FEN-1, originated soon after cells putatively evolved. While reverse transcriptase and topoisomerase I, Topo I, appeared late in the pre-divergence era, when the genetic code was essentially complete. The transition from RNA genes to a DNA genome seemingly proceeded via formation of a DNA-RNA heteroduplex. These results suggest formation of DNA awaited evolution of a catalyst with a hydrophobic domain, capable of sequestering radical bearing intermediates in its synthesis from ribonucleotide precursors. Late formation of topology altering protein, Topo I, further suggests consolidation of genes into chromosomes followed synthesis of comparatively thermostable DNA strands.
...
PMID:Molecular evolution before the origin of species. 1222 77

The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse-transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt-1) in both synaptosomes and GPVs. Uptake of [3H]D-aspartate or [3H]L-glutamate into these preparations revealed sodium-dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine-o-sulfate, l-trans-2,4-pyrrolidine dicarboxylate (PDC) (+/-)-threo-3-methylglutamate and (2S,4R )-4-methylglutamate. The IC50 values found for these compounds suggested functional expression of the 'glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 micro m dihydrokainate, failed to unmask any functional non-EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.
...
PMID:The 'glial' glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate uptake into adult rodent nerve endings. 1255 72

The influence of activation of glutamate receptor (GluR) on outward K(+) current in cultured neonate rat hippocampal astrocytes was investigated. Patch-clamp analysis of K(+) channel currents in cultured astrocytes identified the existence of 71 +/- 6 and 161 +/- 11 pS single-channel K(+) currents that were sensitive to changes in voltage and [Ca(2+)](i) and blocked by external TEA but not by charybdotoxin, iberiotoxin, apamin, or 4-aminopyridine. Reverse transcriptase (RT)-PCR and Northern blot analysis revealed transcripts of the Ca(2+)-activated K(+) channel (K(Ca)) beta(4)-subunit (beta4) (KCNMB4) in cultured astrocytes. Expression of the metabotropic glutamate receptor (mGluR) subtypes mGluR1 and mGluR5 and the ionotropic glutamate receptor (iGluR) subtypes iGluR1 and iGluR4 were detected by RT-PCR and immunofluorescence analysis in cultured astrocytes. The mGluR agonists L-glutamate and quisqualate increased the open state probability (NP(o)) of the 71 and 161 pS K(+) channel currents that were prevented by the mGluR receptor antagonists 1-aminoindan-1,5-dicarboxylic acid or L-(+)-2-amino-3-phosphonopropionic acid and not by the iGluR antagonists (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate or CNQX. Activation of the two types of K(+) channel currents by mGluR agonists was attenuated by pertussis toxin and by inhibition of phospholipase C (PLC) or cytochrome P450 arachidonate epoxygenase. These results indicate that brain astrocytes contain the KCNMB4 transcript and express two novel types of K(Ca) channels that are gated by activation of a G-protein coupled metabotropic glutamate receptor functionally linked to PLC and cytochrome P450 arachidonate epoxygenase activity.
...
PMID:Metabotropic glutamate receptor activation enhances the activities of two types of Ca2+-activated k+ channels in rat hippocampal astrocytes. 1262 72

Identification of the regulatory inputs that direct megakaryocytopoiesis and platelet production is essential for the development of novel therapeutic strategies for the treatment of thrombosis and related hematologic disorders. We have previously shown that primary human megakaryocytes express the N-methyl-d-aspartate acid (NMDA) receptor 1 (NR1) subunit of NMDA-type glutamate receptors, which appear to be pharmacologically similar to those identified at neuronal synapses, responsible for mediating excitatory neurotransmission in the central nervous system. However, the functional role of NMDA receptor signaling in megakaryocytopoiesis remains unclear. Here we provide evidence that demonstrates the fundamental importance of this signaling pathway during human megakaryocyte maturation in vitro. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of RNA extracted from CD34+-derived megakaryocytes identified expression of NR2A and NR2D receptor subunits in these cells, as well as the NMDA receptor accessory proteins, Yotiao and postsynaptic density protein 95 (PSD-95). In functional studies, addition of a selective NMDA receptor antagonist, MK-801 inhibited proplatelet formation, without affecting proliferation or apoptosis. Exposure of CD34+ cells to MK-801 cultured for 14 days in the presence of thrombopoietin induced a decrease in expression of the megakaryocyte cell surface markers CD61, CD41a, and CD42a compared with controls. At an ultrastructural level, MK-801-treated cells lacked alpha-granules, demarcated membranes, and multilobed nuclei, which were prominent in untreated mature megakaryocyte controls. Using immunohistochemistry on sections of whole tibiae from c-Mpl knockout mice we demonstrated that megakaryocytic NMDA receptor expression was maintained following c-Mpl ablation. These data support a fundamental role for glutamate signaling in megakaryocytopoiesis and platelet production, which is likely to be independent of thrombopoietin-mediated effects.
...
PMID:NMDA receptor-mediated regulation of human megakaryocytopoiesis. 1264 30

The expression of gadA and gadB, which encode two glutamate decarboxylases (GADs) of Escherichia coli, is induced by an acidic environment and participate in acid resistance. In this study, we constructed a polyamine-deficient mutant and investigated the role of polyamines in acid resistance. The expression of gadA and gadB was shown to be dependent on polyamines. For that reason, the polyamine-deficient mutant was completely devoid of GAD activity and was very susceptible to low pH if large amounts of polyamines were not provided. We also showed that the polyamine-deficient mutant contained higher cAMP levels than the isogenic polyamine-proficient wild type, and cAMP negatively regulated the expression of gadA and gadB. Therefore, introduction of the cya (encoding adenylate cyclase) mutation allele into the polyamine-deficient mutant resulted in the increment of GAD activity and thus restored the reduced acid resistance of the mutant. The positive regulators, H-NS (histone-like protein, encoded by the hns gene) and RpoS (alternative RNA polymerase sigma subunit, encoded by rpoS gene), also significantly governed the expression of gadA and gadB, respectively. However, polyamines did not regulate either the intracellular H-NS level or rpoS expression under these culture conditions. These results strongly suggest that there are at least two different regulatory systems in acid resistance, one is positive regulation via a H-NS/RpoS system and the other is negative regulation via a polyamine/cAMP system.
...
PMID:Polyamines and glutamate decarboxylase-based acid resistance in Escherichia coli. 1267 Sep 30


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---