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 high affinity branched-chain amino acid transport system (LIV-I) in Pseudomonas aeruginosa is composed of five components: BraC, a periplasmic binding protein for branched-chain amino acids; BraD and BraE, integral membrane proteins; BraF and BraG, putative nucleotide-binding proteins. By using a T7 RNA polymerase/promoter system we overproduced the BraD, BraE, BraF, and BraG proteins in Escherichia coli. The proteins were found to form a complex in the E. coli membrane and solubilized from the membrane with octyl glucoside. The LIV-I transport system was reconstituted into proteoliposomes from solubilized proteins by a detergent dilution procedure. In this reconstituted system, leucine transport was completely dependent on the presence of all five Bra components and on ATP loaded internally to the proteoliposomes. Alanine and threonine in addition to branched-chain amino acids were transported by the proteoliposomes, reflecting the substrate specificity of the BraC protein. GTP replaced ATP well as an energy source, and CTP and UTP also replaced ATP partially. Consumption of loaded ATP and concomitant production of orthophosphate were observed only when BraC and leucine, a substrate for LIV-I, were added together to the proteoliposomes, indicating that the LIV-I transport system has an ATPase activity coupled to translocation of branched-chain amino acids across the membrane.
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PMID:Solubilization and reconstitution of the Pseudomonas aeruginosa high affinity branched-chain amino acid transport system. 140 Apr 43

The exopolysaccharide alginate is a major virulence factor of Pseudomonas aeruginosa strains that infect the lungs of cystic fibrosis patients. The synthesis of alginate is almost uniquely associated with the pathogenicity of P. aeruginosa within the environment of the cystic fibrosis lung. The gene algC is one of the essential alginate biosynthetic genes and codes for the enzyme phosphomannomutase. In this report, we present data on the transcriptional regulation of algC expression. The activity of the algC promoter is modulated by the response regulator, AlgR1, a member of the two-component signal transduction protein family, which also regulates other alginate-specific promoters. In both mucoid (alginate-positive) and nonmucoid (alginate-negative) P. aeruginosa strains, transcriptional activation of algC increased with the osmolarity of the culture medium. This osmolarity-induced activation was found to be dependent on AlgR1. AlgR1 was found to interact directly with the algC promoter. Deletion mapping, in conjunction with mobility shift assays, showed that AlgR1 specifically bound with two regions of algC upstream DNA. A fragment spanning nucleotide positions -378 to -73 showed strong specific binding, while a fragment located between positions -73 and +187 interacted relatively weakly with AlgR1. Phosphorylation of the AlgR1 protein resulted in the stimulation of its in vitro ability to bind to the algC promoter region (a fragment spanning nucleotides -378 to -73). Transcription from the algC promoter, which has significant homology with the RNA polymerase sigma-54 (RpoN) recognition sequence, decreased in an rpoN mutant of P. aeruginosa.
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PMID:Alginate synthesis in Pseudomonas aeruginosa: environmental regulation of the algC promoter. 144 38

The gene product of braB encoding the Na+(Li+)-coupled carrier protein for L-leucine, L-isoleucine, and L-valine (LIV-II carrier) of Pseudomonas aeruginosa PML strain was identified and overexpressed using a T7 RNA polymerase/promoter plasmid system. The gene product was pulse-labeled with [35S]methionine as a protein of an apparent Mr of 34,000 on a sodium dodecyl sulfate-polyacrylamide gel. Cell membranes overproducing the LIV-II carrier were solubilized with n-dodecyl beta-D-maltopyranoside. The carrier protein was purified from the detergent extract by two purification steps: (i) immunoaffinity column chromatography using purified polyclonal antibody directed against synthetic 13-mer peptide corresponding to the carboxyl terminus region of the carrier and (ii) subsequent DEAE-cellulose column chromatography. The detergent was replaced by n-octyl beta-D-glucopyranoside prior to the first elution and phospholipid was present during purification. Proteoliposomes reconstituted with the purified LIV-II carrier exhibited Na+ or Li+ concentration gradient-driven transport of leucine, isoleucine, and valine. These results show that the LIV-II carrier was purified to be in a functional form.
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PMID:Immunoaffinity purification and reconstitution of sodium-coupled branched-chain amino acid carrier of Pseudomonas aeruginosa. 154 99

In order to better understand the regulation of Pseudomonas aeruginosa flagellin expression we cloned the sigma factor of RNA polymerase used to transcribe the flagellin gene. It is a member of the sigma 28 class of alternative sigma factors described in several bacterial genera. Using the published sequence of the fliA gene encoding the sigma 28 from Salmonella typhimurium, we designed two oligonucleotides and, using the polymerase chain reaction, isolated the fliA gene from S. typhimurium chromosomal DNA. This heterologous probe was used in the DNA blot analysis of restriction digests of P. aeruginosa DNA. A 1.7 kb SalI-EcoRI fragment reacted with the probe and this fragment was cloned into the pBluescript vectors. The P. aeruginosa fliA gene was able to complement the motility defect of an Escherichia coli fliA mutant, but only when transcription was driven from the vector promoter. Insertional inactivation of the fliA gene with a gentamicin gene cassette rendered P. aeruginosa nonmotile and unable to express the flagellin gene. The 1.7 kb cloned fragment was sequenced and shown to contain the entire fliA gene. P. aeruginosa FliA shares 67% amino acid similarity with the homologous S. typhimurium sequence. Transcriptional analysis of the fliA gene showed that its expression was not dependent on RpoN, a sigma factor shown also to be required for flagellin synthesis. A reading frame downstream of fliA was found to encode the P. aeruginosa homologue of the enterobacterial cheY gene.
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PMID:The fliA (rpoF) gene of Pseudomonas aeruginosa encodes an alternative sigma factor required for flagellin synthesis. 156 Jul 74

Transcription from the promoter of a positive regulatory gene, xylS, on the TOL plasmid of Pseudomonas putida is activated by another positive regulator, XylR, in the presence of m-xylene and is dependent on RNA polymerase containing the NtrA protein (sigma 54). Deletion analysis of the upstream region of the xylS gene revealed an upstream regulatory sequence (URS), located between 145 and 188 bp upstream from the transcription start site. The URS is active in either orientation and can be placed 3.9 kb further upstream without loss of activity. Dependence of activation on helical periodicity was observed in the region between the URS and the promoter of the xylS gene, suggesting DNA loop formation between these two sites, which are located about 100 bp apart. The expression of xylR was autogenously repressed by XylR protein. This autogenous repression is decreased in an NtrA- background, irrespective of the presence of the xylS promoter in cis, indicating that NtrA protein, or NtrA-containing RNA polymerase that is not bound to the xylS promoter, is involved in the binding of XylR protein to the URS.
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PMID:Analysis of an upstream regulatory sequence required for activation of the regulatory gene xylS in xylene metabolism directed by the TOL plasmid of Pseudomonas putida. 162 97

Escherichia coli RNA polymerases containing mutated alpha subunits were tested for their ability to respond to three different positive regulators (activators) in vitro. The two alpha (rpoA) mutants, alpha-256 and alpha-235, have deletions of the C-terminal 73 and 94 amino acids, respectively. In runoff transcription assays catalyzed by reconstituted holoenzyme, the effects of the mutations on each of three promoters tested were different: activation of the lambda pRM promoter by cI protein (repressor) was nearly normal, activation of the lambda pRE promoter by cII protein was reduced approximately fivefold, and direct activation of the trpPB promoter of Pseudomonas aeruginosa was completely inhibited. We also found that the reconstituted mutant enzyme was defective in recognition of trpPI in the absence of activator. The differential responses of the three promoters to their activators in the presence of the mutant enzymes indicate that the location of an activator-binding site does not by itself determine the region of RNA polymerase with which the activator interacts.
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PMID:Activation defects caused by mutations in Escherichia coli rpoA are promoter specific. 162 75

Mutants which are defective in catabolite repression control (CRC) of multiple independently regulated catabolic pathways have been previously described. The mutations were mapped at 11 min on the Pseudomonas aeruginosa chromosome and designated crc. This report describes the cloning of a gene which restores normal CRC to these Crc- mutants in trans. The gene expressing this CRC activity was subcloned on a 2-kb piece of DNA. When this 2-kb fragment was placed in a plasmid behind a phage T7 promoter and transcribed by T7 RNA polymerase, a soluble protein with a molecular weight (MW) of about 30,000 was produced in Escherichia coli. A soluble protein of identical size was overproduced in a Crc- mutant when it contained the 2-kb fragment on a multicopy plasmid. This protein could not be detected in the mutant containing the vector without the 2-kb insert or with no plasmid. When a 0.3-kb AccI fragment was removed from the crc gene and replaced with a kanamycin resistance cassette, the interrupted crc gene no longer restored CRC to the mutant, and the mutant containing the interrupted gene no longer overproduced the 30,000-MW protein. Pools of intracellular cyclic AMP and the activities of adenylate cyclase and phosphodiesterase were measured in mutant and wild-type strains with and without a plasmid containing the crc gene. No consistent differences between any strains were found in any case. These results provide original evidence for a 30,000-MW protein encoded by crc+ that is required for wild-type CRC in P. aeruginosa and confirms earlier reports that the mode of CRC is cyclic AMP independent in this bacterium.
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PMID:Cloning of a catabolite repression control (crc) gene from Pseudomonas aeruginosa, expression of the gene in Escherichia coli, and identification of the gene product in Pseudomonas aeruginosa. 165 83

Tagetitoxin, a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. tagetis, inhibits RNA synthesis directed by chloroplast RNA polymerase. In isolated chloroplasts, tagetitoxin quickly and specifically reduced the incorporation of [3H]uridine into RNA. When it was added to transcriptionally active chloroplast protein extracts, the toxin directly inhibited incorporation of [32P]UTP into RNA. In addition, tagetitoxin inhibited in vitro RNA synthesis directed by the RNA polymerase from Escherichia coli. In vitro transcription reactions directed by chloroplast RNA polymerase or E. coli RNA polymerase are inhibited at tagetitoxin concentrations less than 1 microM. Nuclear RNA polymerase II purified from wheat germ was only affected at tagetitoxin concentrations greater than 100 microM during in vitro transcription. Tagetitoxin concentrations as high as 1 mM did not affect in vitro transcription reactions directed by RNA polymerase from bacteriophage T7 or SP6.
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PMID:Tagetitoxin inhibits RNA synthesis directed by RNA polymerases from chloroplasts and Escherichia coli. 168 34

In vitro, Pseudomonas aeruginosa TrpI protein activates transcription initiation at the trpBA promoter (trpPB) and represses initiation at its own promoter (trpPI), which diverges from, and overlaps, trpPB. Indoleglycerol phosphate (InGP) reduces the TrpI concentration required for binding to its strong binding site (site I), as measured by repression of trpPI; it also facilitates activation of trpPB, presumably because it enables TrpI to bind to a weaker binding site (site II) and thereby interact with RNA polymerase. The role of site II and InGP in regulation of the two promoters was investigated by constructing site II mutants. A 2 bp substitution affected the ability of TrpI to activate trpPB, but did not significantly affect TrpI binding to site II. A more extensive (8 bp) substitution inhibited TrpI-mediated activation of trpPB and TrpI-mediated protection of site II in a DNase I footprinting assay. However, the mutation did not alter the pattern of TrpI binding observed in gel retardation experiments. In particular, a more slowly-migrating complex (Complex 2) whose appearance was correlated with TrpI binding to site II was formed equally well on a wild-type or substituted DNA fragment. Based on the mutant phenotypes, we propose that a particular sequence of protein--protein and protein--DNA interactions is required for activation of trpPB by TrpI and InGP.
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PMID:Mutations in TrpI binding site II that differentially affect activation of the trpBA promoter of Pseudomonas aeruginosa. 175 20

Transposons such as bacteriophage Mu provide a means to clone bacterial genes as alternatives to using standard recombinant DNA technologies. A DNA-cloning and gene-expressing system has been developed with a bacteriophage Mu (DNA capacity of 38 kb) vector that combines the Mu transposition capabilities and a specialized promoter from bacteriophage T7. Genes cloned with this vector can be identified by transcription in vivo with T7 RNA polymerase and subsequent host translation. This system, illustrated with the characterization of a 35-kb region of the Escherichia coli K-12 chromosome, is applicable to other Enterobacteriaceae, which are hosts for Mu phage, and is potentially applicable to other bacteria, including Pseudomonas aeruginosa, which have Mu-like phage, and to other organisms for which high-frequency transposons are available.
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PMID:Genome mapping and protein coding region identification using bacteriophage Mu. 182 84


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