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 gene encoding the principal sigma factor from Synechococcus sp. strain PCC 7002 was isolated and characterized. The Synechococcus sp. strain PCC 7002 sigA gene encodes a protein of 375 amino acids (43 center dot 7 kDa) that is required for viability under normal growth conditions. The SigA protein was overproduced in Escherichia coli and the purified protein was used to raise polyclonal antiserum in rabbits. This antiserum was used in immunoblot analyses of partially purified RNA polymerase from Synechococcus sp. strain PR6000. The probable in vivo translational start site was identified by a comparison of amino acid sequencing results obtained with SigA proteins overproduced in E. coli with immunoblot analyses of SigA protein in crude preparations of RNA polymerase from the cyanobacterium. The sigA gene is encoded on a transcript of 1700 bases that initiates 496 nucleotides upstream from the probable in vivo translational start site. The abundance of sigA transcripts decreases rapidly after the removal of combined nitrogen from the growth medium.
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PMID:The sigA gene encoding the major sigma factor of RNA polymerase from the marine cyanobacterium Synechococcus sp. strain PCC 7002: cloning and characterization. 893 8

We performed molecular characterization of the RpoD1 protein encoded by the rpoD1 gene isolated from a cyanobacterium, Microcystis aeruginosa K-81. The deduced amino acid sequence (416 aa, 48,871 Da) of RpoD1 exhibited extensive similarity to those of proteins of the eubacterial RpoD family (Escherichia coli sigma 70 homologs). We overproduced and purified RpoD1 (54 kDa) from E. coli. Biological and biochemical analyses suggested that RpoD1 has a function homologous to that of E. coli sigma 70 as follows: (i) the RpoD1 protein complemented an rpoD mutant of E. coli strain YN543 (rpoD285) and (ii) the heterologous RNA polymerase holoenzyme reconstituted from the E. coli core enzyme and recombinant RpoD1 was specifically transcribed from E. coli promoters. Furthermore, Western blot analysis with antiserum against Synechococcus sp. strain PCC 7942 RpoD1 (a principal sigma factor of the sigma 70 type) indicated that M. aeruginosa K-81 RpoD1 (sigma A1) is the principal sigma factor, which is a major component of the sigma subunit on exponential cell growth.
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PMID:The rpoD1 gene product is a principal sigma factor of RNA polymerase in Microcystis aeruginosa K-81. 894 37

The Rieske 2Fe-2S protein is a distinguishing subunit of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for overproduction in Escherichia coli of fusion, full-length, and truncated forms of the Rieske (PetC) protein from the cyanobacterium Nostoc sp. PCC 7906. A glutathione S-transferase/Rieske fusion protein was used to prepare specific chicken egg-yolk antibodies against the Rieske protein. Expression of the nonfusion petC gene in a T7 RNA polymerase promoter vector produced copious quantities of the full-length Rieske protein predominantly as inclusion bodies. The highly enriched, Rieske protein from inclusion bodies has been denatured in guanidine hydrochloride and refolded and the characteristic 2Fe-2S cluster reconstituted in vitro by incubation with iron and sulfide under reducing conditions. Purification by chromatography on Whatman DE52 cellulose and ultrafiltration through a 30000 molecular weight cutoff membrane yielded pure and predominantly monomeric Rieske protein. Reconstituted Rieske preparations showed intense and highly characteristic gx = 1.74, gy = 1.89, and gz = 2.03 "Rieske-type" electron paramagnetic resonance signals at 15 K. Two methods of reconstitution yielded Rieske preparations in which 20-60% of the protein contained 2Fe-2S clusters as determined by EPR spin quantitation. The reconstituted Rieske protein was soluble and stable at 4 degrees C in buffers containing nonionic detergents and showed a redox midpoint potential of +321 mV at pH 7.0 as determined by optical circular dichroism (CD) spectroscopy. These data demonstrate the in vitro restoration of a Cys and His liganded 2Fe-2S cluster and provide the basis for mutational and structural analysis of a PetC Rieske protein of oxygenic photosynthesis.
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PMID:Reconstitution of the 2Fe-2S center and g = 1.89 electron paramagnetic resonance signal into overproduced Nostoc sp. PCC 7906 Rieske protein. 895 2

Genes coding for putative chlorophyll a synthase (chlG) from Synechocystis sp. PCC 6803 and bacteriochlorophyll a synthase (bchG) from Rhodobacter capsulatus were amplified by the polymerase chain reaction and cloned into T7 RNA polymerase-based expression plasmids. In vitro enzymatic assays indicated that heterologous expression of the chlG and bchG gene products in Escherichia coli conferred chlorophyll a and bacteriochlorophyll a synthase activity, respectively. Chlorophyll a synthase utilized chlorophyllide a, but not bacteriochlorophyllide a, as a substrate, whereas bacteriochlorophyll a synthase utilized bacteriochlorophyllide a, but not chlorophyllide a. Both enzymes were also observed to exhibit a marked preference for phytyl diphosphate over geranylgeranyl diphosphate.
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PMID:Characterization of chlorophyll a and bacteriochlorophyll a synthases by heterologous expression in Escherichia coli. 909 96

To study the transcriptional apparatus and the mechanisms that control gene expression in cyanobacteria, the RNA polymerase was purified from the filamentous Calothrix sp. PCC 7601 and used in in vitro transcription assays. Conditions required for specific transcription initiation to occur were analyzed with the eleven Calothrix PCC 7601 genes for which the 5' ends have been mapped. Most of the transcripts directly obtained did not have the expected size, providing a test for looking at specific transcription factors. Addition of RcaA, a protein that binds to the promoter region of the phycobiliprotein cpeBA operon, restored accurate initiation of transcription in the in vitro system for three phycobiliprotein promoters. RcaA thus is a transcription factor that allows to mimick in vivo transcription. In parallel, the functional properties of the Escherichia coli and cyanobacterial RNA polymerases were compared. The enteric enzyme could not precisely initiate transcription at the promoter of a phycobiliprotein gene and, reciprocally, the cyanobacterial RNA polymerase could initiate transcription at PlacUV5, but not from wild-type Plac promoters. The different behaviours of the enzymes are discussed in the light of the structural differences that exist between subunits of the RNA polymerases.
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PMID:Promoter recognition by a cyanobacterial RNA polymerase: in vitro studies with the Calothrix sp. PCC 7601 transcriptional factors RcaA and RcaD. 952 97

An 11-year-old boy with hypertension was suspected of having bilateral adrenal pheochromocytomas and hyperplasia. Molecular analysis of specific tumor suppressor genes and oncogenes excluded the familial syndromes, von Hippel-Lindau (VHL) disease and multiple endocrine neoplasia (MEN) type 2A. Further evaluation identified a unilateral adrenal pheochromocytoma with a VHL heterozygous somatic mutation (G695A) and loss of the maternal allele at 11p15.5-11p14 exclusively in the tumor tissue. Both reverse-transcriptase polymerase chain reaction and immunohistochemistry confirmed increased expression of IGF2 within the tumoral tissue, relative to a normal control adrenal gland. These results ruled out familial syndromes and suggested that the VHL mutation and the loss of maternal allele on chromosome 11 could have contributed to tumor development.
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PMID:Molecular characterization of a pediatric pheochromocytoma with suspected bilateral disease. 1117 29

In many cell types, cell death induced by a variety of insults is accompanied by an increase in intracellular calcium. The Ca(2+) homeostatic mechanisms affected by such insults, however, have not been fully determined. Recent evidence indicates that kainic acid-induced seizures alter plasma membrane calcium ATPase mRNA expression within vulnerable hippocampal cell populations before the onset of cell death. We examined the effects of altering plasma membrane calcium ATPase expression on cell vulnerability in rat pheochromocytoma 12 cells. Pheochromocytoma 12 cells are vulnerable to Ca(2+) overload induced by the Ca(2+) ionophore A23187. Reverse transcriptase-PCR and Western blot data indicated that plasma membrane calcium ATPase isoform 4b constitutes a major calcium pump isoform in the pheochromocytoma 12 cells. Therefore, permanently transfected pheochromocytoma 12-derived cell lines were established that either over-expressed plasma membrane calcium ATPase isoform 4b, or suppressed the expression of the endogenous plasma membrane calcium ATPase isoform 4. Over-expressing clones were less vulnerable to Ca(2+)-mediated cell death induced by A23187 whereas "antisense" clones were considerably more susceptible. These data indicate that regulation of plasma membrane calcium ATPase expression may be critical to cellular survival when cells are exposed to pathological increases in intracellular calcium.
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PMID:Plasma membrane calcium ATPase plays a role in reducing Ca(2+)-mediated cytotoxicity in PC12 cells. 1139 91

RNA polymerase was purified from the unicellular cyanobacterium, Synechococcus sp. strain PCC 7942, and found to be associated with a 52 kilodalton (kDa) polypeptide. The determined N-terminal sequence of the polypeptide was identical to the predicted amino-acid sequence of the rpoD1 gene product. Furthermore, the rpoD1 gene is suggested to be indispensable for viability by the inability to disrupt the gene. These results indicate that the rpoD1 gene product is the principal sigma factor of RNA polymerase.
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PMID:The rpoD1 gene of Synechococcus sp. strain PCC 7942 encodes the principal sigma factor of RNA polymerase. 1250 49

The expression of RNA polymerase (RNAP) sigma factor genes and proteins was characterized as a first step toward understanding their functions in a unicellular cyanobacterium Synechocystis sp. PCC 6803, which can perform photosynthesis. All nine sigma factors (group 1, SigA; group 2, SigB to SigE; and group 3, SigF to SigI) and each RNAP core subunit (RpoA, RpoB, RpoC1 and RpoC2) were overproduced and purified from Escherichia coli cells, then polyclonal antibodies were prepared. Western blot and primer extension analyses revealed that the intracellular levels of group 1 and 2 sigma factors ranged from 0.9 fmol to 9.3 fmol per microgram of the total protein under conditions of steady-state growth, and that growth phase-dependent or constitutive transcripts were observed. Interestingly, no group 3 sigma factor proteins were detected under normal physiological conditions whereas their transcripts were robust, implying a possible regulation of translational attenuation and/or protein instability. Phylogenetic analysis also revealed that group 3 sigma factor homologues of cyanobacteria are conserved with evolutionary or functionary divergence among them. In vitro and in vivo results indicated significant evidence of high-light responsive SigD expression and its promoter recognition of the photosynthesis gene, psbA. On the other hand, autoregulated sigB transcription, a dramatically increased SigB expression upon the exposure of cells to heat-shock, and specific promoter recognition by SigB with redundancy of other sigma factors on the heat-shock hspA promoter were observed. These findings clearly indicated that SigB is a heat-shock responsive sigma factor. The unique promoter architecture and expression of the relevant sigma factor gene are also discussed herein.
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PMID:Purification, characterization, and gene expression of all sigma factors of RNA polymerase in a cyanobacterium. 1252 96

Phylogenetic analysis of cyanobacteria was carried out using the small subunit rRNA (16S rRNA), DNA gyrase subunit B (gyrB), DNA-dependent RNA polymerase gamma subunit (rpoC1) and a principal sigma factor of E. coli sigma(70) type for DNA-dependent RNA polymerase (rpoD1) gene sequences of 24 strains which contained 5 subgroups of cyanobacteria-3 strains of the Chroococcales, 5 strains of the Pluerocapsales, 7 strains of the Oscillatoriales, 7 strains of the Nostocales and 2 strains of the Stigonematales. Degenerated PCR primers of gyrB, rpoC1 and rpoD1 genes were designed using consensus amino acid sequences registered in GenBank. The phylogenetic positions of cyanobacteria were resolved through phylogenetic analysis based on 16S rDNA, gyrB, rpoC1 and rpoD1 gene sequences. Phylogenies of gyrB, rpoC1 and rpoD1 support 16S rRNA-based classification of cyanobacteria. Interestingly, phylogenies from amino acid sequences deduced from gyrB and combined amino acid sequences deduced from rpoC1 and rpoD1 genes strongly support that of 16S rRNA, but the branching pattens of the trees based on 16S rDNA, GyrB, rpoC1, rpoD1 and combined amino acid sequences deduced from rpoC1 and rpoD1 were not congruent. In this study, we showed the correlation among phylogenetic relationships of 16S rDNA, gyrB, rpoC1 and rpoD1 genes. The phylogenetic trees based on the sequences of 16S rDNA, GyrB, rpoC1, rpoD1 and the combined amino acid sequences deduced from rpoC1 and rpoD1 showed that the lateral gene transfer of rRNA might be suspected for Synechocystis sp. PCC 6803.
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PMID:The phylogenetic relationships of cyanobacteria inferred from 16S rRNA, gyrB, rpoC1 and rpoD1 gene sequences. 1294


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