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)

Oligosaccharide-plasmid DNA conjugates were synthesized simply and effectively via the diazocoupling method. Plasmids (pUC19, pTRI-beta-actin, and pEGFP-C1) were treated with an N-beta-lactoside-substituted diazonium salt to yield diazocoupling products with degree of substitutions of 2.5-3.1 mol% of overall nucleobases. The lactose-pUC19 conjugate was found to resist restriction enzymes more strongly than the nonconjugated plasmid DNA and to acquire a strong binding affinity to galactose-specific lectin RCA(120). The diazocoupling modification of pTRI-beta-actin plasmid DNA little influenced in vitro transcription with T7 RNA polymerase. When lactose-pEGFP-C1 conjugate was transfected to baby hamster kidney (BHK) cells by means of cationic lipids, transduced gene was expressed in BHK cells similarly with the nonconjugated pEGFP-C1. The modification of plasmid DNA with carbohydrate enhanced the resistance to restriction enzymes and developed a strong binding affinity to galactose-specific lectin.
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PMID:Conjugation of plasmid DNAs with lactose via diazocoupling enhances resistance to restriction enzymes and acquires binding affinity to galactose-specific lectin. 1040 69

Based on the analysis of molecular interactions of proteins with DNA binding sites, a new approach to developing mathematical models describing gene expression is introduced. Detection of hierarchical structures in metabolic networks can be used to decompose complex reaction schemes. This will be achieved by assigning each regulator protein to one level in the hierarchy. Signals are then transduced from the top level to the lower level, but not vice versa. The method is shown by a simple example with two interacting proteins. A comparison of simulation results shows good agreement between a model taking all interactions into account and a model developed with the new approach. Finally, the method is applied to the crpA modulon in Escherichia coli, which controls uptake and metabolism for a number of carbohydrates. Here, RNA polymerase represents the top level, CrpA the second level, and the lactose-specific repressor LacI the lowest level, respectively. Besides the lactose operon, the method is applied to the adenylate cyclase gene and the gene for the regulator CrpA.
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PMID:The organization of metabolic reaction networks. II. Signal processing in hierarchical structured functional units. 1128 90

Osteoclasts or their precursors interact with the glycoprotein-enriched matrix of bone during extravasation from the vasculature, and upon attachment prior to resorption. Reverse transcriptase-PCR studies showed that two new alternatively spliced forms of chicken galectin-3, termed Gal-3TM1 and Gal-3TR1, were enriched and preferentially expressed in highly purified chicken osteoclast-like cells. Gal-3TM1 and Gal-3TR1 mRNA were also detected in chicken intestinal tissue, but not in kidney, liver, or lung. Gal-3TM1 and Gal-3TR1 messages both contain an open reading frame encoding a predicted 70-amino acid TM1 sequence inserted between the N-terminal Gly/Pro repeat domain and the carbohydrate recognition domain (exons 3 and 4). Gal-3TR1 mRNA contains an additional 241-bp sequence, which encodes a truncated open reading frame between the 4th and 5th exons, and, whose translation is expected to terminate within the carbohydrate recognition domain encompassing exons 4, 5, and 6. Immunoblotting and affinity chromatography showed that purified osteoclast preparations and intestinal homogenates contained a 36-kDa lactose-binding galectin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analyses on chymotryptic peptides from the 36-kDa lectin confirmed its identity as Gal-3TM1. The TM1 insert contains a single transmembrane-spanning region and a leucine zipper-like stalk domain that is predicted to position the intact carbohydrate recognition domain of Gal-3TM1 on the exterior surface of the plasma membrane. Immunofluorescent staining of chicken osteoclasts confirmed the expression of Gal-3TM1 at the plasma membrane. Gal-3TM1 is the first example of a galectin superfamily member capable of being expressed as a soluble protein and as a transmembrane protein.
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PMID:New alternatively spliced form of galectin-3, a member of the beta-galactoside-binding animal lectin family, contains a predicted transmembrane-spanning domain and a leucine zipper motif. 1188 49

Transcription by RNA polymerase can stimulate localized DNA supercoiling in Escherichia coli. In vivo, there is extensive experimental support for a "twin-domain" model in which positive DNA supercoils are generated ahead of a translocating RNA polymerase complex and negative supercoils are formed behind it. Negative supercoils accumulate in the template DNA because the positive supercoils are preferentially removed by cellular topoisomerase action. Yet, in vitro, clear and convincing support for the twin-domain mechanism has been lacking. In this article, we reconcile this inconsistency by showing that, in a defined in vitro system with plasmid DNA templates, a variety of sequence-specific DNA-binding proteins, such as the bacteriophage lambda O replication initiator or the E. coli lactose or galactose repressors, strikingly stimulate transcription-coupled DNA supercoiling. We demonstrate further that this stimulation requires the presence in the DNA template of a recognition sequence for the relevant DNA-binding protein and depends on the production of long RNA chains by an RNA polymerase. Our data are most consistent with a model in which specific DNA-binding proteins facilitate a twin-domain mechanism to enhance DNA supercoiling during transcription. More precisely, we suggest that some nucleoprotein complexes, perhaps those that contain sharply bent DNA, can form barriers that impede the diffusion and merger of independent chromosomal supercoil domains. Localization of DNA supercoils by nucleoprotein complexes may serve as a general mechanism for modulating DNA transactions that are sensitive to DNA superhelicity.
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PMID:Potent stimulation of transcription-coupled DNA supercoiling by sequence-specific DNA-binding proteins. 1209 6

A complex of plasmid DNA-lactose conjugate with lectin is proposed as an artificial system to control transcription activity: the in vitro transcription of DNA in the conjugate with T7 RNA polymerase was repressed in the presence of RCA120, and then the transcription ability was recovered by adding lactose or a lactose-carrying polymer to the repression system.
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PMID:Artificial regulation of transcription applying carbohydrate-lectin interaction. 1212 4

The sigma(54) subunit of the RNA polymerase directs the expression of specific operons in association with cognate activators. Three different activators have been detected in the Listeria monocytogenes genome on the basis of the high conservation of a specific domain. Among them, the LacR activator, of the LevR family, was found just upstream from a newly described sigma(54)-dependent operon, lpo, which presents a classical -24/-12 consensus promoter. The lpo operon encodes proteins similar to subunits of a PTS permease (EII) of the lactose family, namely LpoA (IIA) and LpoB (IIB). It also encodes a third putative protein, LpoO, with an unknown function but sharing high similarity with proteins also encoded within PTS operons from other bacteria and bearing a RGD motif. The expression of lpo was clearly dependent on LacR and sigma(54), and was induced by cellobiose, chitobiose and lactose. It underlies that the lpo operon likely encodes proteins involved in the utilization of these sugars by L. monocytogenes.
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PMID:Characterization of a unique sigma54-dependent PTS operon of the lactose family in Listeria monocytogenes. 1450 17

RNA interference (RNAi) is a newly described biological phenomenon mediated by small interfering RNA (siRNA) that targets mRNA for degradation by cellular enzymes and has become a powerful method for studying gene functions in mammalian systems. The development of systems for inducing siRNA expression should enable examination of acute loss-of-function phenotypes in a cell of interest without the need to consider lethality or epigenetic adaptation of cells. We describe in this report an inducible siRNA expression system made by combined utilization of the RNA polymerase III-dependent promoter H1 and the bacterial lac repressor. Using this system, we established AGS gastric epithelial cells in which expression of SHP-2, a cellular tyrosine phosphatase known to specifically bind the Helicobacter pylori virulence factor CagA, is conditionally and reversibly silenced by the lactose analog isopropyl-1-thio-beta-D-galactopyranoside (IPTG). Upon expression in AGS cells, CagA provoked a morphological transformation, termed the hummingbird phenotype, which is associated with CagA virulence. This morphogenetic activity of CagA was totally abolished when SHP-2 expression was silenced by inducible siRNA expression in AGS cells. Our results indicate that SHP-2 is a critical downstream effector of H. pylori CagA. The conditional gene silencing system described here should become a powerful tool for investigating the roles of cancer-related genes through a reversed genetic approach.
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PMID:Conditional gene silencing utilizing the lac repressor reveals a role of SHP-2 in cagA-positive Helicobacter pylori pathogenicity. 1513 73

Inducible expression systems in which T7 RNA polymerase transcribes coding sequences cloned under control of a T7lac promoter efficiently produce a wide variety of proteins in Escherichia coli. Investigation of factors that affect stability, growth, and induction of T7 expression strains in shaking vessels led to the recognition that sporadic, unintended induction of expression in complex media, previously reported by others, is almost certainly caused by small amounts of lactose. Glucose prevents induction by lactose by well-studied mechanisms. Amino acids also inhibit induction by lactose during log-phase growth, and high rates of aeration inhibit induction at low lactose concentrations. These observations, and metabolic balancing of pH, allowed development of reliable non-inducing and auto-inducing media in which batch cultures grow to high densities. Expression strains grown to saturation in non-inducing media retain plasmid and remain fully viable for weeks in the refrigerator, making it easy to prepare many freezer stocks in parallel and use working stocks for an extended period. Auto-induction allows efficient screening of many clones in parallel for expression and solubility, as cultures have only to be inoculated and grown to saturation, and yields of target protein are typically several-fold higher than obtained by conventional IPTG induction. Auto-inducing media have been developed for labeling proteins with selenomethionine, 15N or 13C, and for production of target proteins by arabinose induction of T7 RNA polymerase from the pBAD promoter in BL21-AI. Selenomethionine labeling was equally efficient in the commonly used methionine auxotroph B834(DE3) (found to be metE) or the prototroph BL21(DE3).
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PMID:Protein production by auto-induction in high density shaking cultures. 1591 65

A novel strategy for artificial regulation system of gene expression applying the specific molecular recognition between carbohydrate and lectin is proposed. Plasmid-lactose conjugates (pActin-lactose and pGFP-lactose) prepared via diazocoupling maintained the transcription activity with T7 RNA polymerase. Gel-shift assay showed that the pActin-lactose conjugates were specifically complexed with galactose-specific lectin RCA(120) with a strong binding affinity (K(a) = 7.6 x 10(5) M(-1) per Lac-unit). The complexes were observed to form aggregates of sub-several micrometer size by means of transmission electron microscopy (TEM) and atomic force microscopy (AFM). The activities of transcription and expression of the conjugates were evaluated, respectively, on the basis of the amount of transcript of pActin and the fluorescent intensity of the expressed GFP. These activities were repressed in the presence of an increasing concentration of RCA120, and then recovered by adding lactose, lactosylceramide-containing liposomes, and lactose-carrying polymers to the conjugate-RCA120 complex. Gel-shift assay and TEM observation revealed that the aggregation form of the complex was relaxed partially in the presence of the lactose derivatives, which increased the accessibility of T7 RNA polymerase to result in the recovery of transcription activity.
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PMID:On-off switching of gene expression regulated with carbohydrate-lectin interaction. 1615 90

Guanosine 3',5'-bispyrophosphate (ppGpp), also known as "magic spot," has been shown to bind prokaryotic RNA polymerase to down-regulate ribosome production and increase transcription of amino acid biosynthesis genes during the stringent response to amino acid starvation. Because many environmental growth perturbations cause ppGpp to accumulate, we hypothesize ppGpp to have an overarching role in regulating the genetic program that coordinates transitions between logarithmic growth (feast) and growth arrest (famine). We used the classic glucose-lactose diauxie as an experimental system to investigate the temporal changes in transcription that accompany growth arrest and recovery in wild-type Escherichia coli and in mutants that lack RelA (ppGpp synthetase) and other global regulators, i.e., RpoS and Crp. In particular, diauxie was delayed in the relA mutant and was accompanied by a 15% decrease in the number of carbon sources used and a 3-fold overall decrease in the induction of RpoS and Crp regulon genes. Thus the data significantly expand the previously known role of ppGpp and support a model wherein the ppGpp-dependent redistribution of RNA polymerase across the genome is the driving force behind control of the stringent response, general stress response, and starvation-induced carbon scavenging. Our conceptual model of diauxie describes these global control circuits as dynamic, interconnected, and dependent upon ppGpp for the efficient temporal coordination of gene expression that programs the cell for transitions between feast and famine.
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PMID:Guanosine 3',5'-bispyrophosphate coordinates global gene expression during glucose-lactose diauxie in Escherichia coli. 1646 49


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