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)

Regulation of virulence factor expression in Bordetella pertussis is mediated by the BvgAS two-component regulatory system. Although previous studies have demonstrated that the transcriptional regulation of the filamentous hemagglutinin gene (fhaB) involves binding of the BvgA activator directly to the fhaB promoter region, the mechanism of pertussis toxin operon (ptx) regulation by BvgA has remained unclear. We demonstrate in vitro the specific binding of BvgA to a region upstream of the ptx promoter that encompasses a 20-bp directly repeated sequence (positions -157 to -117) previously shown to be critical for BvgA-dependent activation. This binding is strictly dependent on the phosphorylation of BvgA, which can be obtained by incubation of BvgA with acetyl phosphate. By DNase I protection studies, we demonstrate the synergistic binding of BvgA-phosphate and purified Escherichia coli RNA polymerase to the ptx promoter. In the presence of the polymerase holoenzyme, a greatly extended footprint encompassing the region between -163 and the putative polymerase binding site was observed. The implications of these observations for pertussis toxin expression and regulation are discussed.
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PMID:Synergistic binding of RNA polymerase and BvgA phosphate to the pertussis toxin promoter of Bordetella pertussis. 759 24

Studies were performed to determine the mechanism underlying deficient arginine vasopressin (AVP)-stimulated adenylyl cyclase activity in chronic renal failure (CRF). As compared to control, principal cells cultured from the inner medullary collecting tubule of rats previously made uremic by 5/6 nephrectomy fail to accumulate cAMP when stimulated with AVP. CRF cells do respond normally to forskolin or cholera toxin and the defect in AVP responsiveness is not prevented by treatment with pertussis toxin, by cyclooxygenase inhibition, or by inhibition or down-regulation of protein kinase C. In contrast to their lack of responsiveness to AVP, CRF cells respond normally to other agonists of adenylyl cyclase such as isoproterenol or prostaglandin E2. Plasma membranes prepared from the inner medullae of CRF rats exhibit a marked decrease in apparent AVP receptor number but no change in the apparent number of beta adrenergic receptors. Reverse transcriptase PCR of total RNA from the inner medullae of CRF animals reveals virtual absence of V2 receptor mRNA; mRNA for alpha s is present in normal abundance. These studies indicate that AVP resistance in CRF is due, at least in part, to selective down-regulation of the V2 receptor as a consequence of decreased V2 receptor mRNA.
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PMID:Vasopressin resistance in chronic renal failure. Evidence for the role of decreased V2 receptor mRNA. 761 8

cDNA species encoding either the long or the short isoforms of the rat thyrotropin-releasing-hormone (TRH) receptor were expressed stably in Rat 1 fibroblasts, and clones expressing specific binding of [3H]TRH were detected and expanded. Clones expressing each of these receptors at levels up to 1 pmol/mg of membrane protein were selected for analysis. Reverse-transcriptase PCR on RNA isolated from these clones confirmed that each clone expressed only mRNA corresponding to the expected splice variant. Both receptor splice variants bound [3H]TRH with a Kd of some 80 nM when binding assays were performed in the presence of guanosine 5'-[beta gamma-imido]-triphosphate. In the presence of TRH, both receptor subtypes were able to cause stimulation of inositol phosphate generation in a pertussis-toxin-insensitive manner with similar EC50 values and to stimulate the mobilization of intracellular Ca2+, but, despite reports that TRH receptors can also interact with the G-proteins Gs and Gi2, neither receptor splice variant was able to modulate adenylate cyclase activity in either a positive or a negative manner. These data indicate that the long and short isoforms of the rat TRH receptor have similar affinities for TRH and display similar abilities to interact with the Gq-like G-proteins, but show no ability to regulate adenylate cyclase, at least when expressed in this genetic background.
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PMID:Comparison of the signalling properties of the long and short isoforms of the rat thyrotropin-releasing-hormone receptor following expression in rat 1 fibroblasts. 764 58

Using a combination of polymerase chain reaction and genomic library screening we have cloned a human gene for a subtype of the somatostatin (SST) receptor (SSTR) termed human SSTR5 (hSSTR5), which is located on chromosome 16. The predicted amino acid sequence of hSSTR5 displays 75% sequence identity with a recently identified rat SSTR [Mol. Pharmacol. 42:939-946 (1992)], suggesting that it is the human homologue of this receptor. hSSTR5 consists of a 363-residue polypeptide exhibiting a putative seven-transmembrane domain topology typical of G protein-coupled receptors. The receptor displays considerable sequence identity to hSSTR1 (42%), hSSTR2 (48%), hSSTR3 (47%), and hSSTR4 (46%). Membranes prepared from COS-7 cells transiently expressing the hSSTR5 gene bound 125I-Leu8,D-Trp22,Tyr25-SST-28 (125I-LTT-SST-28) with high affinity and in a saturable manner. SST-14, SST-28, and various synthetic SST peptide agonists produced dose-dependent inhibition of radioligand binding with the following rank order of potency: LTT-SST-28 > SST-28 > D-Trp8-SST-14 > SST-14 approximately RC-160 approximately BIM 23014 > MK-678 > SMS 201-995. hSSTR5 bound SST-28 with a 12.6-fold greater affinity (Ki = 0.19 nM), compared with SST-14 (Ki = 2.24 nM), indicating that the receptor is SST-28 selective. Addition of GTP, guanosine-5'-O-(3-thio)triphosphate, Na+ ions, or pertusis toxin greatly reduced 125I-LTT-SST-28 binding, thereby indicating that hSSTR5 is coupled to pertussis toxin-sensitive G proteins. Both SST-14 and SST-28 displayed dose-dependent inhibition of forskolin-stimulated cAMP accumulation, consistent with functional coupling of the receptor to adenylyl cyclase inhibition. Northern blot analysis of SSTR5 mRNA revealed a 2.4-kilobase transcript in normal rat pituitary and GH3 rat pituitary tumor cells and a 4.0-kilobase transcript in normal human pituitary. Reverse transcriptase polymerase chain reaction revealed expression of the hSSTR gene in fetal human pituitary and hypothalamus but not in human cerebral cortex. In situ hybridization of the rat pituitary showed that SSTR5 mRNA is selectively localized in the anterior lobe. SSTR5 mRNA was not expressed in four human pituitary tumors (somatotroph adenoma, prolactinoma, and chromophobe adenomas) or in a human insulinoma. Although hSSTR5 displays approximately 75% sequence identity with rat SSTR5, the two receptors display significantly different pharmacological profiles, especially with respect to their binding affinities for the SST analogue SMS 201-995.
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PMID:Molecular cloning, functional characterization, and chromosomal localization of a human somatostatin receptor (somatostatin receptor type 5) with preferential affinity for somatostatin-28. 790 5

In Bordetella pertussis, expression of virulence factors is controlled by the Bvg proteins, which comprise a sensor-regulator two-component signal transduction system. Previously, we described a mutant strain of B. pertussis that had reduced transcription of pertussis toxin and adenylate cyclase toxin genes, while other virulence factors were relatively unaffected. We obtained a B. pertussis clone that repaired the defect in both this strain and an independent mutant strain with a similar phenotype when introduced onto the chromosome by allelic exchange. Further analysis revealed that the mutations were just upstream of the translational start site of the rpoA gene encoding the alpha subunit of RNA polymerase. We confirmed that these mutations were responsible for the mutant phenotype by site-directed mutagenesis. Our hypothesis that these mutations cause an overexpression of rpoA was confirmed by Western immunoblotting and translational fusion analysis. Corroboration of this effect was obtained by overexpressing rpoA on a plasmid in wild-type B. pertussis, which caused the same phenotype as the mutants showed. Conclusions in regard to the identity of the transcription activator of the toxin genes are discussed.
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PMID:Effect of mutations causing overexpression of RNA polymerase alpha subunit on regulation of virulence factors in Bordetella pertussis. 796 98

The class II IL-8 receptor (IL-8R) binds both melanoma growth stimulatory activity (MGSA) and IL-8 with high affinity. Reverse transcriptase polymerase chain reaction demonstrates that the class II IL-8R mRNA, which has previously been detected only in cells of hematopoietic lineage, is also expressed in non-hematopoietic cell types shown to respond to MGSA or IL-8. To study the signaling mechanism by MGSA through the class II IL-8R in non-hematopoietic cells, this receptor was overexpressed in the 3ASubE human placental and the 293 human kidney cell lines. Membrane preparations of the class II IL-8R expressing 3ASubE transfectants exhibited a 2.3 +/- 0.2-fold increase in GTP gamma 35S binding, which was sensitive to pertussis toxin, in response to MGSA treatment (0.2 microM). This MGSA response was not observed in cells transfected with the parental expression vector. In vivo phosphorylation studies demonstrated that the class II IL-8R was basally phosphorylated in the untreated transfectants, and MGSA (5 nM) treatment markedly enhanced the phosphorylation of this receptor. The MGSA-induced receptor phosphorylation was both time and concentration dependent and could be mimicked by treatment with the calcium ionophore A23187. Phosphoamino acid analysis indicated that the MGSA-induced receptor phosphorylation was on serine residue(s), suggesting that a serine kinase is activated in response to MGSA binding to the class II IL-8R in non-hematopoietic cells.
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PMID:Melanoma growth stimulatory activity enhances the phosphorylation of the class II interleukin-8 receptor in non-hematopoietic cells. 829 49

The bvg locus of Bordetella pertussis encodes an environmentally inducible operon essential for the expression of virulence genes. We show that in Escherichia coli, the PTOX promoter cloned in cis of the bvg locus is activated and environmentally regulated. Cotransformation of E. coli with the bvg locus cloned in a low-copy-number plasmid and with the PTOX promoter cloned in a high-copy-number plasmid can give rise to two different results. If the PTOX promoter is cloned in the pGem-3 vector, transcription is absent. If the PTOX promoter is cloned in the plasmid pKK232, containing the PTOX promoter between two ribosomal gene terminators of transcription, transcription occurs, although regulation of transcription is abolished. Under these conditions, the intracellular amount of RNA transcripts is increased by adding to the culture medium novobiocin, an inhibitor of bacterial gyrases. In vitro, the transcription of the PTOX promoter is activated on E. coli RNA polymerase supplemented with cell extracts from wild-type B. pertussis. Addition of DNA gyrase to the mixture dramatically reduces the amount of RNA synthesized. Our data show that the products of the bvg locus, BvgA and BvgS, are directly involved in the regulation of the PTOX promoter in E. coli and that DNA topology may play a role in the induction of transcription.
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PMID:DNA topology affects transcriptional regulation of the pertussis toxin gene of Bordetella pertussis in Escherichia coli and in vitro. 839 6

The cyaA gene of Bordetella pertussis and of Bordetella bronchiseptica encodes a toxin which is a bifunctional protein exhibiting adenylate cyclase and haemolytic activities. In Bordetella, virulence factors are synthesized under the control of the bvg regulatory locus, in response to environmental signals. In Escherichia coli the cyaA gene is not expressed, nor is it activated by bvg indicating that the activation of cya by bvg is indirect. To characterize cis-acting regulatory regions required for the activation of the cyaA gene we constructed cyaA-lacZY fusions containing progressive deletions in the promoter upstream region and isolated promoter mutations by chemical and site-directed mutagenesis. Deletion analysis shows that a region extending from -569 to -136 bp upstream from the start site of transcription is required for transactivation by bvg, suggesting that multiple binding sites are involved in the activation of the cyaA promoter. No single or double mutations in the promoter upstream region were found which conferred inactive or bvg-independent Cya phenotype. A double mutation in positions +10 and +13, relative to the transcription start site, rendered the promoter bvg-independent and functional in E. coli. The constitutive mutations create a new transcription start site, 20 bp downstream from the wild-type site, by providing new -10 and -35 elements recognized by RNA polymerase alone.
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PMID:Functional analysis of the cya promoter of Bordetella pertussis. 846 14

In Bordetella pertussis the expression of virulence factors is coordinately regulated by the BvgS and BvgA proteins, members of the bacterial two-component signal transduction family, BvgS being the transmembrane sensor and BvgA the regulator. Activation of virulence gene expression requires phosphorylation of BvgA. On the basis of observed differences in the regulation of individual genes, the existence of accessory regulators has been postulated. They were supposed to be necessary for expression of genes encoding adenylate cyclase toxin (cya) and pertussis toxin (ptx), but not required for the expression of fha, encoding filamentous hemagglutinin. To clarify this issue we investigated the mechanism of activation of the BvgAS-controlled genes by performing in vitro run-off transcription experiments. We show, using purified RNA polymerase of B.pertussis, that phosphorylated BvgA is sufficient for transcriptional activation of the major virulence genes, thus providing good evidence that BvgA regulation operates directly with the transcription initiation machinery at the promoters of the virulence genes without a requirement for accessory activators. In addition, our results indicate that activation of the different promoters may involve distinct mechanisms. We suggest that the previously observed differences in regulation of individual virulence-associated genes reflect differences in the phosphorylation state of BvgA.
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PMID:Phosphorylated BvgA is sufficient for transcriptional activation of virulence-regulated genes in Bordetella pertussis. 859 92

Recently, a novel type of regulatory mutation causing differential effects on the expression of virulence genes due to a slight overexpression of the RNA polymerase alpha subunit (RpoA) was found in Bordetella pertussis (N. H. Carbonetti, T. M. Fuchs, A. A. Patamawenu, T. J. Irish, H. Deppisch, and R. Gross, J. Bacteriol. 176:7267-7273, 1994). To gather information on the molecular events behind this phenomenon, we isolated suppressor mutants of the RpoA-overexpressing strains after random mutagenesis. Genetic characterization of these suppressor strains revealed the existence of at least three distinct groups of dominant alleles. Mutations occurred either in the rpoA locus itself, in the bvg locus, or in unknown gene loci. One mutant of the latter group was further characterized. By the introduction of a cosmid library containing genomic B. pertussis DNA into this suppressor strain, we isolated a cosmid which suppressed the phenotype of the suppressor strain, thus restoring the negative effect on transcription of the ptx and cya toxin genes. Mutagenesis of the cosmid with Tn5 led to the identification of the gene locus responsible for this phenomenon. Its DNA sequence revealed the presence of an open reading frame (ORF) consisting of 2,373 bp coding for a hypothetical 86-kDa protein with extensive sequence similarities to ORFs with not yet identified functions of Escherichia coli, Haemophilus influenzae, and Neisseria meningitidis. The new gene, termed tex, for toxin expression, seems to be an essential factor for B. pertussis, as it cannot be deleted from the bacterial chromosome. All members of this new protein family show significant sequence similarities with the mannitol repressor protein MtlR and with the presumptive RNA-binding domains of the Pnp and ribosomal S1 proteins of E. coli in their N- and C-terminal parts, respectively. These sequence similarities and the fact that the tex gene was isolated by virtue of its effects on gene expression in B. pertussis indicate that the members of this new protein family may play an important role in the transcription machinery of prokaryotic organisms.
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PMID:A new gene locus of Bordetella pertussis defines a novel family of prokaryotic transcriptional accessory proteins. 875 71


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