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 expression of six novel genes located in the region from abrB to spoVC of the Bacillus subtilis chromosome was analyzed, and one of the genes, yabG, had a predicted promoter sequence conserved among SigK-dependent genes. Northern blot analysis revealed that yabG mRNA was first detected from 4 h after the cessation of logarithmic growth (T(4)) in wild-type cells and in a gerE36 (GerE(-)) mutant but not in spoIIAC (SigF(-)), spoIIGAB (SigE(-)), spoIIIG (SigG(-)), and spoIVCB (SigK(-)) mutants. The transcription start point was determined by primer extension analysis; the -10 and -35 regions are very similar to the consensus sequences recognized by SigK-containing RNA polymerase. Inactivation of the yabG gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of yabG spores in L-alanine and in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride was also the same as that of wild-type spores. On the other hand, the protein preparation from yabG spores included 15-, 18-, 21-, 23-, 31-, 45-, and 55-kDa polypeptides which were low in or not extracted from wild-type spores under the same conditions. We determined their N-terminal amino acid sequence and found that these polypeptides were CotT, YeeK, YxeE, CotF, YrbA (31 and 45 kDa), and SpoIVA, respectively. The fluorescence of YabG-green fluorescent protein fusion produced in sporulating cells was detectable in the forespores but not in the mother cell compartment under fluorescence microscopy. These results indicate that yabG encodes a sporulation-specific protein which is involved in coat protein composition in B. subtilis.
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PMID:The Bacillus subtilis yabG gene is transcribed by SigK RNA polymerase during sporulation, and yabG mutant spores have altered coat protein composition. 1071 92

Tandem pore domain acid-sensitive K(+) channel 3 (TASK-3) is a new member of the tandem pore domain potassium channel family. A cDNA encoding a 365- amino acid polypeptide with four putative transmembrane segments and two pore regions was isolated from guinea pig brain. An orthologous sequence was cloned from a human genomic library. Although TASK-3 is 62% identical to TASK-1, the cytosolic C-terminal sequence is only weakly conserved. Analysis of the gene structure identified an intron within the conserved GYG motif of the first pore region. Reverse transcriptase-polymerase chain reaction analysis showed strong expression in brain but very weak mRNA levels in other tissues. Cell-attached patch-clamp recordings of TASK-3 expressed in HEK293 cells showed that the single channel current-voltage relation was inwardly rectifying, and open probability increased markedly with depolarization. Removal of external divalent cations increased the mean single channel current measured at -100 mV from -2.3 to -5.8 pA. Expression of TASK-3 in Xenopus oocytes revealed an outwardly rectifying K(+) current that was strongly decreased in the presence of lower extracellular pH. Substitution of the histidine residue His-98 by asparagine or tyrosine abolished pH sensitivity. This histidine, which is located at the outer part of the pore adjacent to the selectivity filter, may be an essential component of the extracellular pH sensor.
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PMID:TASK-3, a novel tandem pore domain acid-sensitive K+ channel. An extracellular histiding as pH sensor. 1074 66

The expression of gal operon in Escherichia coli is driven by two promoters, P1 and P2 separated by 5 bp. The transcription initiated from the P2 generates a large amount of abortive transcripts to produce a comparable amount of full-length transcript as P1 in vitro. In this study, we investigated the source of the abortive transcripts by employing a quantitative potassium permanganate footprinting method that determines the extent of open promoter complex formation. The extents of open promoter complex formation at the two gal promoters were about the same during the given reaction time while the amount of transcription initiation determined by in vitro transcription assay showed a considerable difference: several hundred-fold more transcription initiation from the P2 than the P1, most of which was abortive. Thus, it was concluded that the abortive transcripts are generated reiteratively by a small fraction of RNA polymerase. An in vitro transcription assay using an immobilized DNA template revealed that the fraction of RNA polymerase generating abortive transcripts never produces the full-length transcript and it remains bound to the promoter. We concluded that there are two kinds of RNA polymerase-promoter complexes formed at galP2, at least in vitro, productive complex and nonproductive complex; and, the nonproductive complex is responsible for generating large amount of abortive transcripts from the P2.
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PMID:Reiterative transcription initiation from galP2 promoter of Escherichia coli. 1076 May 80

Humans are exposed occupationally and environmentally to metal aerosols including lead (Pb2+) and cadmium (Cd2+). These toxicants accumulate in male reproductive organs. Epidemiological studies have been equivocal about effects of Pb2+ and Cd2+ on hormone concentrations, male fertility and sperm parameters. Comparison of Pb2+ and Cd2+ concentrations in fertile and infertile men are problematic. Problem areas include failure to control confounding variables, but genetic polymorphisms as in somatic diseases may modulate Pb2+ and Cd2+ damage. Multiple calcium (Ca2+) and potassium (K+) channel isoforms have been identified in human testes and spermatozoa. These Ca2+ and K+ channels are involved in early events of acrosome reactions. Ca2+ channel are susceptible to Cd2+ poisoning and K+ channels to Pb2+. These channels offer entry paths for metallic toxicants into mature spermatozoa. Ion channel polymorphisms may cause differential sensitivities to Cd2+ and Pb2+, explaining in part prospective blinded studies showing high Cd2+ in varicocele-related human infertility and high Pb2+ in unexplained infertility. In both forms of male infertility the ability to undergo an acrosome reaction decreases. Reverse transcriptase-polymerase chain reaction assays for Ca2+ and K+ channel isoforms may identify susceptibility subgroups with lower resistance to environmental exposures.
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PMID:Male infertility and environmental exposure to lead and cadmium. 1078 69

To terminate transcription in E. coli, Rho protein binds an RNA loading site on the nascent transcript, translocates 5'--> 3' along the RNA in an ATP-driven process, and, upon reaching the transcription elongation complex, brings about RNA release. Thus, the Rho-dependent termination process can be viewed, in part, as a kinetic competition between the rate of transcript elongation by RNA polymerase (RNAP) and the rate of Rho translocation along the nascent transcript. In the context of this model, NusG, which is an essential E. coli protein, regulates Rho-dependent termination in an apparently paradoxical way, increasing the rate of transcription elongation of E. coli RNAP in the absence of Rho while also shifting the sites of Rho-dependent termination upstream on the template. Here we investigate the regulation of Rho-dependent termination by NusG. Analytical ultracentrifugation was used to establish the existence of a stable complex of NusG and Rho and to demonstrate a stoichiometry of one NusG monomer per Rho hexamer. Surface plasmon resonance was used to examine the kinetics of the formation and dissociation of the NusG-Rho complex, yielding an association rate constant (k(on)) of 2.8 (+/-0.8) x 10(5) M(-)(1) s(-)(1), a dissociation rate constant (k(off)) of 3.9 (+/-0.7) x 10(-)(3) s(-)(1), and a calculated equilibrium (dissociation) constant (K(d)) of 1.5 (+/-0.3) x 10(-)(8) M. The apparent stability of the NusG-Rho complex is insensitive to changes in salt (potassium acetate) concentration between 0.05 and 0.15 M. The translocation and transcription termination activities of Rho at saturating NusG concentrations were, however, both sensitive to salt concentration over this range, suggesting that these activities do not directly reflect the stability of the NusG-Rho complex. Rho-dependent termination could be demonstrated for transcription complexes in which E. coli RNAP had been substituted by either bacteriophage SP6 or T7 RNAP. NusG, however, was not active in transcription termination assays with either of these phage RNAPs. Thus, we conclude that NusG modulates Rho-dependent termination by interacting specifically with the RNAP of the E. coli elongation complex to render the complex more susceptible to the termination activity of Rho.
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PMID:Regulation of rho-dependent transcription termination by NusG is specific to the Escherichia coli elongation complex. 1082 31

The full-length cDNAs of two different alpha-subunits (Kir6.1 and Kir6.2) and partial cDNAs of three different beta-subunits (SUR1, SUR2A and SUR2B) of ATP-sensitive potassium (KATP) channels of the guinea-pig (gp) were obtained by screening a cDNA library from the ventricle of guinea-pig heart. Cell-specific reverse-transcriptase PCR with gene-specific intron-spanning primers showed that gpKir6.1, gpKir6.2 and gpSUR2B were expressed in a purified fraction of capillary endothelial cells. In cardiomyocytes, gpKir6.1, gpKir6.2, gpSUR1 and gpSUR2A were detected. Patch-clamp measurements were carried out in isolated capillary fragments consisting of 3-15 endothelial cells. The membrane capacitance measured in the whole-cell mode was 19.9 +/- 1.0 pF and was independent of the length of the capillary fragment, which suggests that the endothelial cells were not electrically coupled under our experimental conditions. The perforated-patch technique was used to measure the steady-state current-voltage relation of capillary endothelial cells. Application of K+ channel openers (rilmakalim or diazoxide) or metabolic inhibition (250 microM 2,4-dinitrophenol plus 10 mM deoxyglucose) induced a current that reversed near the calculated K+ equilibrium potential. Rilmakalim (1 microM), diazoxide (300 microM) and metabolic inhibition increased the slope conductance measured at -55 mV by a factor of 9.0 (+/-1.8), 2.5 (+/-0.2) and 3.9 (+/-1.7), respectively. The effects were reversed by glibenclamide (1 microM). Our results suggest that capillary endothelial cells from guinea-pig heart express KATP channels composed of SUR2B and Kir6.1 and/or Kir6.2 subunits. The hyperpolarization elicited by the opening of KATP channels may lead to an increase in free cytosolic Ca2+, and thus modulate the synthesis of NO and the permeability of the capillary wall.
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PMID:ATP-sensitive potassium channels in capillaries isolated from guinea-pig heart. 1083 35

The effect of sepsis on the ubiquitously expressed ATP-sensitive potassium (uK(ATP)-1) channel expression was measured in Sprague-Dawley rat diaphragms. Rats were treated with either 0.5 ml saline or 20 mg/Kg E. coli lipopolysaccharides and sacrificed at 3, 6, 12, 24, or 48 h later. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that channel mRNA expression was increased at 3 h and continued to rise up to 48 h. Western blotting analysis showed a approximately 9-fold increase in channel protein expression 24 h after sepsis. Our results demonstrate that sepsis upregulates the uK(ATP)-1 channel.
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PMID:Induction of the ATP-sensitive potassium (uK(ATP)-1) channel by endotoxemia. 1084 76

Ribosomal RNA transcription initiation requires the melting of DNA to form an open complex, formation of the first few phosphodiester bonds, commencement of RNA polymerase I movement along the DNA, clearance of the promoter, and the formation of a steady-state ternary elongation complex. We examined DNA melting and promoter clearance by using potassium permanganate, diethylpyrocarbonate and methidiumpropylEDTA.Fe(II) footprinting. In combination, these methods demonstrated: (1) TIF-IB and RNA polymerase I are the only proteins required for formation of an initial approximately 9 base-pair open promoter region. This finding contradicts earlier results using diethylpyrocarbonate alone, which suggested an RNA synthesis requirement for stable melting. (2) DNA melting is temperature-dependent, with a tm between 15 and 20 degrees C. (3) Temperature-dependency of melting, as well as stalling the polymerase at sites close to the transcription start site revealed that the melted DNA region initially opens upstream of the transcription initiation site, and enlarges in a downstream direction coordinate with initiation, eventually attaining a steady-state transcription bubble of approximately 19 base-pairs. (4) The RNA-DNA hybrid protects the template DNA from single-strand footprinting reagents. The hybrid is 9 bp in length, consistent with the longer hybrid estimated by some for the Escherichia coli polymerase and with the hybrids estimated for eukaryotic polymerases II and III.
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PMID:DNA melting and promoter clearance by eukaryotic RNA polymerase I. 1086 Jul 23

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

Synaptotagmins (Syt) play important roles in Ca(2+)-induced neuroexocytosis. Insulin secretion of the pancreatic beta-cell is dependent on an increase in intracellular Ca(2+); however, Syt involvement in insulin exocytosis is poorly understood. Reverse transcriptase-polymerase chain reaction studies showed the presence of Syt isoforms III, IV, V, and VII in rat pancreatic islets, whereas Syt isoforms I, II, III, IV, V, VII, and VIII were present in insulin-secreting betaTC3 cell. Syt III and VII proteins were identified in rat islets and betaTC3 and RINm5F beta-cells by immunoblotting. Confocal microscopy showed that Syt III and VII co-localized with insulin-containing secretory granules. Two-fold overexpression of Syt III in RINm5F beta-cell (Syt III cell) was achieved by stable transfection, which conferred greater Ca(2+) sensitivity for exocytosis, and resulted in increased insulin secretion. Glyceraldehyde + carbachol-induced insulin secretion in Syt III cells was 2.5-fold higher than control empty vector cells, whereas potassium-induced secretion was 6-fold higher. In permeabilized Syt III cells, Ca(2+)-induced and mastoparan-induced insulin secretion was also increased. In Syt VII-overexpressing RINm5F beta-cells, there was amplification of carbachol-induced insulin secretion in intact cells and of Ca(2+)-induced and mastoparan-induced insulin secretion in permeabilized cells. In conclusion, Syt III/VII are located in insulin-containing secretory granules, and we suggest that Syt III/VII may be the Ca(2+) sensor or one of the Ca(2+) sensors for insulin exocytosis of the beta-cell.
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PMID:Synaptotagmin III/VII isoforms mediate Ca2+-induced insulin secretion in pancreatic islet beta -cells. 1093 83


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