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 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 complete nucleotide sequence of a 3.2-kilobase-pair chromosomal region containing the algP and algQ genes was determined. The algQ gene encodes an acidic 18-kilodalton polypeptide required for transcriptional activation of the algD gene. The algD gene product catalyzes a critical step in alginate biosynthesis, and its overproduction is necessary for the emergence of mucoid Pseudomonas aeruginosa during chronic infections in cystic fibrosis. A novel genetic element, algP, was identified immediately downstream of algQ. This gene appears to act synergistically with algQ. Unlike a biosynthetic gene, algD, and another regulatory gene, algR, which undergo transcriptional activation in mucoid cells, both algP and algQ are equally transcribed in mucoid and nonmucoid isogenic strains of P. aeruginosa. The promoter regions of algP and algQ were mapped by using S1 nuclease protection analysis. The algQ promoter was also analyzed and showed activity in an in vitro transcriptional runoff assay with major RNA polymerase species from P. aeruginosa and Escherichia coli. The putative algQ and algP promoter sequences, unlike algD and algR, resemble sigma 70-utilized promoters from E. coli and appeared constitutively transcribed at a low level in P. aeruginosa. The algP gene has an unusual DNA sequence, with multiple direct repeats organized in six highly conserved, tandemly arranged, 75-base-pair (bp) units. At a lower level, this sequence had 45 degenerate repeats of 12 bp overlapping with the 75-bp repeats and extending beyond the region of 75-bp repeats. The algP repeats appeared important for the function of the algQ-algP regulatory region in maintaining mucoidy.
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PMID:DNA sequence and expression analysis of algP and algQ, components of the multigene system transcriptionally regulating mucoidy in Pseudomonas aeruginosa: algP contains multiple direct repeats. 211 Jan 44

The triggering of mucoidy (formation of the exopolysaccharide alginate) in Pseudomonas aeruginosa is accomplished primarily in Cystic Fibrosis lung environment through activation of the promoter of a gene algD, which encodes GDP-mannose dehydrogenase, by the product of a regulatory gene algR1. Both algD and algR1 promoter regions have significant homology, including the presence of sequences recognized by the RNA polymerase sigma 54. We demonstrate that the algR1 promoter is partly constitutive and its activation, similar to that of algD, is dependent on high osmolarity as well as the presence of its own gene product and is repressed by high concentrations of AlgR1. The RpoN sigma factor also plays a critical role in the transcription of both algD and algR1 genes.
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PMID:Control of alginate synthesis in Pseudomonas aeruginosa: regulation of the algR1 gene. 251 Jul 18

To investigate myeloid cell maturation, we established a panel of monoclonal antibodies that recognize myeloid cell nuclear antigens. One of these monoclonal antibodies was used to purify a specific protein complex (PC) from a human spleen. This PC, which is present at high levels in peripheral blood monocytes and granulocytes, contains a protein that is the cystic fibrosis (CF) antigen. The purified PC was shown to inhibit the activity of casein kinase I and II but not cAMP-dependent protein kinase, protein kinase C, v-abl tyrosine kinase, or insulin receptor tyrosine kinase. The observed Ki values for casein kinases I and II purified from several sources were 1 microM or less. Furthermore, the addition of the purified PC to a nuclear extract from human cells was able to prevent protein kinase-mediated stimulation of RNA polymerase activity. The unique inhibitory character of the PC and its elevated levels in monocytes and granulocytes and of the CF antigen in CF patients implies that this complex may be associated with myeloid cell functions and perhaps with the cause or consequence of the clinical manifestations of CF.
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PMID:A protein containing the cystic fibrosis antigen is an inhibitor of protein kinases. 265 77

Chronic infection by alginate-producing (mucoid) Pseudomonas aeruginosa is the leading cause of mortality among cystic fibrosis (CF) patients. During the course of sustained infection, the production of an alginate capsule protects the bacteria and allows them to persist in the CF lung. One of the key regulators of alginate synthesis is the algT (algU) gene encoding a putative alternative sigma factor (sigma E). AlgT was hyperproduced and purified from Escherichia coli. The N-terminal sequence of the purified protein matched perfectly with that predicted from the DNA sequence. The purified protein, in the presence of E. coli RNA polymerase core enzyme, was able to initiate transcription of an algT promoter. Deletion of the -35 region of this promoter abolished this activity in vitro as well as in vivo. These data indicate that the algT gene encodes a sigma factor that is autoregulatory.
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PMID:The algT (algU) gene of Pseudomonas aeruginosa, a key regulator involved in alginate biosynthesis, encodes an alternative sigma factor (sigma E). 764 17

The mucoid phenotype is common among strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis and is due to overproduction of an exopolysaccharide called alginate. However, the mucoid phenotype is unstable in vitro, especially when the cells are incubated under low oxygen tension. Spontaneous conversion to the nonmucoid form is typically due to mutations (previously called algS) that are closely linked to the alginate regulatory gene algT, located at 68 min on the chromosome. Our sequence analysis of algT showed that its 22-kDa gene product shares homology with several alternate sigma factors in bacteria, suggesting that AlgT (also known as AlgU) interacts directly with RNA polymerase core to activate the promoters of alginate genes. AlgT showed striking sequence similarity (79%) to sigma E of Escherichia coli, an alternate sigma factor involved in high-temperature gene expression. Our analysis of the molecular basis for spontaneous conversion from mucoid to nonmucoid, in the cystic fibrosis isolate FRD, revealed that nonmucoid conversion was often due to one of two distinct missense mutations in algT that occurred at codons 18 and 29. RNase protection assays showed that spontaneous nonmucoid strains with the algT18 and algT29 alleles have a four- to fivefold reduction in the accumulation of algT transcripts compared with the wild-type mucoid strain. Likewise, a plasmid-borne algT-cat transcriptional fusion was about 3-fold less active in the algT18 and algT29 backgrounds compared with the mucoid wild-type strain, and it was 20-fold less active in an algT::Tn501 background. These data indicate that algT is autoregulated. The spontaneous algT missense alleles also caused about fivefold-reduced expression of the adjacent negative regulator, algN (also known as mucB). Transcripts of algN were essentially absent in the algT::Tn501 strain. Thus, algT regulates the algTN cluster, and the two genes may be cotranscribed. A primer extension analysis showed that algT transcription starts 54 bp upstream of the start of translation. Although the algT promoter showed little similarity to promoters recognized by the vegetative sigma factor, it was similar to the algR promoter. This finding suggests that AlgT may function as a sigma factor to activate its own promoter and those of other alginate genes. The primer extension analysis also showed that algT transcripts were readily detectable in the typical nonmucoid strain PAO1, which was in contrast to a weak signal seen in the algT18 mutant of FRD. A plasmid-borne algT gene in PAO1 resulted in both the mucoid phenotype and high levels of algT transcripts, further supporting the hypothesis that AlgT controls its own gene expression and expression of genes of the alginate regulon.
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PMID:Mucoid-to-nonmucoid conversion in alginate-producing Pseudomonas aeruginosa often results from spontaneous mutations in algT, encoding a putative alternate sigma factor, and shows evidence for autoregulation. 796 21

Na+ and Cl- conductances in the apical membrane of respiratory epithelial cells are essential for electrolyte and water transport in the airways. Apart from the well described defect in adenosine 3' : 5' cyclic monophosphate-(cAMP-) dependent activation of Cl- conductances in cystic fibrosis (CF), an increased Na+ conductance has also been reported from transepithelial measurements. In the present experiments we tried to identify these conductances in nasal epithelial cells using patch-clamp and microelectrode techniques. With these methods we found identical and relatively low membrane voltages of about -36 mV in both freshly isolated and primary cultured normal and CF nasal epithelial cells. A Cl- conductance could be activated by cAMP in normal (deltaG = 0.3 +/- 0.8 nS, n = 10) but not in CF (deltaG = 0.3 +/- 0.1 nS, n = 11) cells, whereas Ca2+-dependent Cl- currents activated by adenosine 5'-triphosphate (ATP) and bradykinin were present in both types of cells. Cell-attached membrane patches from stimulated cells did not reveal discernible single-channel events when activated with any of the agonists. A Na+ conductance was also detected in freshly isolated ciliated respiratory cells in impalement studies, as evidenced by the hyperpolarization induced by 10 micromol/l amiloride (deltaV = -5.2 +/- 0.6 mV, n = 56) and when Na+ was replaced in the bath by N-methyl-D-glucamine (NMDG) (deltaV = -5.7 +/- 0.9 mV, n = 14). In whole-cell patch-clamp experiments, the amiloride-induced hyperpolarization was significantly larger in CF (deltaV = 9.7 +/- 2.4 mV, n = 22) when compared to normal (deltaV = -3.3 +/- 0.9 mV, n = 27) cells in short-term culture. Reverse transcriptase polymerase chain reaction analysis of normal respiratory cells identified messenger RNA of both the cystic fibrosis transmembrane conductance regulator (CFTR) as well as the human epithelial Na+ channel (hNaCh). The present experiments confirm the absence of a cAMP-dependent Cl- conductance in CF respiratory epithelial cells and support previous findings obtained in transepithelial and microelectrode studies which indicate an increased Na+ conductance in respiratory epithelial cells from CF patients.
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PMID:Na+ and Cl- conductances in airway epithelial cells: increased Na+ conductance in cystic fibrosis. 858 4

Transcriptional activation of the Pseudomonas aeruginosa algD gene results in high-level synthesis of the capsular polysaccharide alginate, an important P. aeruginosa virulence factor expressed in cystic fibrosis (CF) patients with chronic pulmonary disease. In this study, electrophoretic mobility-shift assays were used to identify a novel protein (AlgZ), which binds specifically to a sequence located 280 bp upstream of the algD promoter. While AlgZ-binding activity did not require the response regulators AlgB or AlgR, expression of AlgZ was found to be absolutely dependent on the alternative sigma factor AlgT. Electrophoretic mobility-shift assays and copper-phenanthroline footprinting localized AlgZ binding to a 36 bp algD region, which includes several helical repeats. A collection of alginate-producing (mucoid) and non-mucoid P. aeruginosa strains, derived from CF patients, was characterized for AlgZ-binding activity. In all cases, AlgZ binding to algD sequences was observed when extracts derived from mucoid P. aeruginosa CF isolates were examined. However, this binding activity was not present when extracts from non-mucoid P. aeruginosa CF isolates were tested. Oligonucleotide mutagenesis was employed to create an algD allele with a 4 bp mutation in the predicted AlgZ-binding site (algD38) and a heterologous substitution allele (algD40), in which the entire AlgZ-binding site was replaced with a non-specific DNA sequence of identical size. When the algD38 mutation was cloned into an algD-cat transcriptional fusion, this resulted in a 28-fold reduction in algD expression, whereas the algD40 mutation abolished algD transcription, indicating that AlgZ acts as an activator of algD transcription. These results support the hypothesis that activation of algD involves the formation of a high-order looped structure allowing for multivalent contacts between AlgZ, AlgR and RNA polymerase containing the alternative sigma factor AlgT. Characterization of the molecular details of algD activation will provide insights into the control of other prokaryotic and eukaryotic promoters that utilize multiple activators.
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PMID:Identification and characterization of AlgZ, an AlgT-dependent DNA-binding protein required for Pseudomonas aeruginosa algD transcription. 889 12

The splicing variant, 5T allele, in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene was shown to be associated with partial penetrance of the clinical expression. This splicing variant leads to two possible transcripts: one normal and the other aberrantly spliced that lacks exon 9. The aim of this study was to analyze the molecular basis of the partial penetrance in individuals carrying the 5T allele. We analyzed the level of the correctly spliced RNA transcribed from the 5T allele in nasal and epididymal epithelium and correlated it with disease expression. Semiquantitative nondifferential reverse-transcriptase-PCR showed a considerable variability (6%-37%) in the total level of correctly spliced RNA transcribed from the 5T allele in nasal epithelium from 11 patients. A significant nonlinear correlation (r = .82, P = .002) between the level of the normal CFTR transcripts and the severity of lung disease was shown. No individuals with normal lung function and minimal or no lung disease (FEV1 >80% predicted) had <25% of normal transcripts, and individuals with <15% of normal transcripts did not have FEV1 >80%. The level of normal transcripts in epididymal epithelial cells from four infertile males with congenital bilateral absence of the vas deferens was low (6%-24%). In infertile males with normal lung function the level of correctly spliced transcripts in the nasal epithelium was higher than the level in the epididymal epithelium. These results indicate that there is variability in the efficiency of the splicing mechanism, among different individuals and between different organs of the same individual. This variability provides the molecular basis of the partial penetrance of cystic fibrosis disease in patients carrying the 5T allele.
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PMID:The molecular basis of partial penetrance of splicing mutations in cystic fibrosis. 898 51

Conversion of Pseudomonas aeruginosa to the mucoid phenotype plays a major role in the pathogenesis of respiratory infections in cystic fibrosis (CF). One mechanism responsible for mucoidy is based on mutations that inactivate the anti-sigma factor, MucA, which normally inhibits the alternative sigma factor, AIgU. The loss of MucA allows AIgU to freely direct transcription of the genes responsible for the production of the exopolysaccharide alginate resulting in mucoid colony morphology. In Escherichia coli, a close homologue of AIgU, sigma(E), directs transcription of several genes under conditions of extreme heat shock. Here we examined whether AIgU, besides its role in controlling alginate production, affects the heat-shock response in P. aeruginosa. The P. aeruginosa rpoH gene encoding a homologue of the major heat-shock sigma factor, sigma32, was found to be transcribed by AIgU containing RNA polymerase from one of its promoters (P3) identified in this study. Transcription of rpoH from P3 was elevated upon exposure to extreme heat shock in an aIgU-dependent manner. Importantly, the AIgU-dependent promoter of rpoH was found to be activated in mucoid mucA mutants. In keeping with this observation, introduction of a wild-type mucA gene abrogated AIgU-dependent rpoH transcription in mucoid P. aeruginosa laboratory isolates and CF isolates. These results suggest that conversion to mucoidy and the heat-shock response are co-ordinately regulated in P. aeruginosa. The simultaneous activation of both systems in mucA mutants, selected in the lungs of CF patients, may have significance for the inflammatory processes characteristic of the establishment of chronic infection and ensuing clinical deterioration in CF.
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PMID:Microbial pathogenesis in cystic fibrosis: co-ordinate regulation of heat-shock response and conversion to mucoidy in Pseudomonas aeruginosa. 915 26


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