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)

We describe a bacterial RNA polymerase mutation, rif 501, which confers rifampicin resistance and thermosensitivity to E. coli K 12. The purified RNA polymerase enzyme from rif 501 bacteria shows increased heatsensitivity in vitro at 51 degrees C. However, in vivo, at 42 degrees C the non-permissive temperature, mutant bacteria continue to grow and to synthesize RNA for 90 min. On a lawn of the mutant bacteria, at 40-41 degrees C, phage lambda forms clear plaques (LycA phenotype); this is probably due to an enhancement of cro function; we surmise that at 42 degrees C the transcription originating from the pR (but not from the pL) promoter on the lamdba genome becomes N-independent and less sensitive to the absence of the cro product. We discuss the possibility that both the N and cro proteins of phage lambda interact directly with the bacterial RNA polymerase. These observations indicate that the loss of viability of the rif 501 mutant at the restrictive temperature is not a consequence of an immediate inactivation of RNA polymerase; rather we feel it is due to a modification of the activity of RNA polymerase, leading to a disruption of the cellular regulation.
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PMID:A bacterial RNA polymerase mutant that renders lambda growth independent of the N and cro functions at 42 degrees C. 77 9

Chromosomal non-histone proteins are obtained from nuclei of two types of pigeon erythroid cells: erythroblasts (cells active in RNA synthesis) and erythrocytes (cells with repressed RNA synthesis). They are well soluble in solutions of low ionic strength. Electrophoretic separation of the obtained non-histone proteins in polyacrylamide gels with urea and SDS shows the presence of qualitative differences in the pattern of non-histone proteins of chromatine from erythroblasts and erythrocytes. By electrophoresis in urea some protein bands of non-histone proteins of chromatine from erythroblasts were found which disappear with the aging of cells. At the same time two protein fractions were observed in chromatine from erythrocytes which were absent in that of erythroblasts. Disappearance of some high molecular weight protein fractions from erythrocyte chromatine as compared to erythroblasts was observed by separation of the non-histone proteins in the presence of SDS. These fractions of the non-histone proteins disappearing during aging of cells are well extractable from erythroblast chromatine by 0.35 M NaCl solution. In the in vitro system with E. coli RNA polymerase addition of non-histone proteins of chromatine from erythroblasts to chromatine from erythrocytes increases RNA synthesis 2--3 times. At the same time addition of non-histone proteins from erythrocytes is either without any influence on this process or somewhat inhibiting.
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PMID:[Comparative investigation of the non-histone proteins of chromatin from pigeon erythroblasts and erythrocytes]. 80 71

Crude calf thymus DNA-dependent RNA polymerase, RNA polymerase B (ribonucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6), was incubated with the tritium labeled, potent inhibitor [3H]amanin, in order to form the enzymatically inactive [3H]amanin-polymerase complex ([3H]A-P complex). Subsequent purification procedures for the [3H]A-P complex were based on radioactive assays. Phosphocellulose chromatography separated two radioactive components: PCI, the previously reported amatoxin binding protein, ABP (Brodner and Wieland, 1976), and PC II, the [3H]A-P complex. Sodium dodecyl sulfate gel electrophoresis of the complex showed the presence of a new heavy band very close to subunit B 1 and a decreased intensity of subunit band B 3. These were the only differences noted in the subunit structure of RNA polymerase B. [3H]Amanin was covalently coupled to the enzyme, affinity labeling, by a water-soluble carbodiimide and the resultant conjugate submitted to sodium dodecyl sulfate gel electrophoresis. The profile of radioactivity showed one main peak (greater than 2000 cpm) coinciding with the 550-nm absorption peak of subunit B 3 on a stained parallel gel. Since no other protein band contains any significant radioactivity, the binding site for [3H]amanin and most probably for all amatoxins is localized on the B 3 subunit SB 3.
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PMID:Identification of the amatoxin-binding subunit of RNA polymerase B by affinity labeling experiments. Subunit B3-the true amatoxin receptor protein of multiple RNA polymerase B. 95 70

The loosely bound chromatin proteins of Ehrlich ascites hyperdiploid cells have been prepared by extraction of chromatin with 0.35 M NaCl. Sodium dodecyl sulfate gel electrophoresis of the 0.35 M NaCl-soluble chromatin proteins reveals high heterogeneity with a molecular weight range of 10,000 to 170,000. The 0.35 M NaCl-soluble chromatin proteins contain many components similar to the more tightly bound non-histone chromatin proteins complex with the loosely bound chromatin proteins by gradient dialysis, the inhibitory effect of histones on transcription of DNA in vitro was reduced. The reconstituted complex manifested a level of template activity similar to that of native chromatin as measured in an Ehrlich ascites tumor RNA polymerase reaction. The loosely bound chromatin proteins contain RNA as well as phosphoproteins. Phenol extraction or DNA affinity chromatography of these proteins yielded fractions enhanced 25- to 30-fold in phosphorus which were capable of stimulating DNA-templated RNA synthesis in vitro. The stimulation of transcription from DNA was template-specific, effective only with a DNA template prepared from Ehrlich ascites tumor, but not from rat liver, calf thymus, or chicken erythrocytes. In addition, the stimulatory effect of the specific DNA-binding proteins appears to be RNA polymerase-specific, the stimulation being manifested with Ehrlich ascites tumor nucleoplasmic RNA polymerase and not with Micrococcus luteus RNA polymerase. Thus, the loosely bound chromosomal proteins from Ehrlich ascites tumor contain a fraction that specifically binds to Ehrlich ascites tumor DNA and exhibits a template- and RNA polymerase-specific stimulatory effect on transcription from DNA.
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PMID:Study of the loosely bound non-histone chromatin proteins. Stimulation of deoxyribonucleic acid-templated ribonucleic acid synthesis by a specific deoxyribonucleic acid-binding phosphoprotein fraction. 111 17

Adult diarrhea rotavirus (ADRV) has caused epidemics of diarrhea in China since 1982 and remains the only group B rotavirus associated with widespread disease in humans. We recently characterized the proteins of ADRV and have now proceeded to identify the gene segments encoding each protein. Viral RNA transcripts were synthesized in vitro with the endogenous viral RNA polymerase and separated by electrophoresis in agarose. The individual transcripts were translated in a cell-free system using nuclease-treated rabbit reticulocyte lysates. The translation products were compared with polypeptides found in purified virus and were characterized by SDS-PAGE, immunoprecipitation, and Western blot analysis using antisera to double- and single-shelled virions, virus cores, and monoclonal antibodies. Furthermore, individual RNA transcripts were hybridized to total dsRNA to determine their genomic origin. Based on this analysis, the core polypeptides VP1, VP2 and VP3 are encoded by segments 1, 2, and 3, respectively. The main polypeptides in the inner capsid, VP6, and the outer capsid, VP4 and VP7, are encoded by segments 6, 4, and 8 respectively. Segments 5, 7, and 9 code for 60, 45, and 30 kDa nonstructural polypeptides. Two other nonstructural polypeptides (24 and 25 kDa) are derived from gene segment 11. Gene segment 10 codes for a 26 kDa polypeptide that is precipitated with serum to ADRV and may be a structural protein VP9. With this exception, gene coding assignments of ADRV are comparable to those of the group A rotaviruses. Our results have clear implications for further work in cloning, sequencing, and expression genes of ADRV and can provide direction towards understanding the origin and the evolution of this virus.
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PMID:Coding assignments of the genome of adult diarrhea rotavirus. 132 59

Three open reading frames (ORFs) have been found in the region downstream of the luxG gene in the Photobacterium leiognathi lux operon. These genes (ORF I, II, and III) are not only closely linked to the lux operon and transcribed in the same direction but also show the same organization and code for proteins homologous in sequence to the gene products of ribB, ribA, and ribH of Bacillus subtilis, respectively. The Photobacterium leiognathi gene (ORF II) corresponding to ribA was expressed in Escherichia coli in the bacteriophage T7 promoter-RNA polymerase system and a 40 kDa 35S-labeled polypeptide has been detected on SDS-PAGE. Expression of DNA extending from luxBEG to ORF II inserted between a strong promoter and a reporter gene and transferred by conjugation into Vibrio harveyi did not affect the expression of the reporter gene. The results provide evidence that neither promoter nor terminator sites were present in the DNA between the luxG and ORF II indicating that these genes might be part of the lux operon.
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PMID:The lux genes in Photobacterium leiognathi are closely linked with genes corresponding in sequence to riboflavin synthesis genes. 133 74

The promoter region of the agarase gene (dagA) of Streptomyces coelicolor A3(2) is complex; it consists of four distinct promoters with different -10 and -35 regions. We report the isolation of a form of RNA polymerase that mediates transcription in vitro from the dagAp4 promoter. The core components of this RNA polymerase are associated with a polypeptide of c. 66 kDa; holoenzyme reconstitution experiments show that the 66 kDa polypeptide functions as a sigma factor that directs transcription from the dagAp4 and Bacillus subtilis veg promoters in vitro. Alignment of the DNA sequences of these two promoters shows that they have bases in common in the -10 and -35 regions and that these sequences are similar to those observed for the major RNA polymerases of other bacteria. N-terminal amino acid sequence analysis of the 66 kDa polypeptide revealed it to be the product of the hrdB gene. Previous experiments showed that the predicted amino acid sequence of the hrdB gene product is very similar to the major sigma factors of other bacteria and suggested that disruption of the hrdB gene is lethal. These observations together lead to the conclusion that we have isolated the major RNA polymerase of Streptomyces coelicolor A3(2). We have developed an improved protocol for the renaturation of sigma factors that have been isolated by preparative sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). This method involves renaturing the polypeptide in the presence of the bacterial chaperonin GroEL. We expect this protocol to find general application for renaturation of other polypeptides that have been subjected to SDS-PAGE.
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PMID:Isolation and characterization of the major vegetative RNA polymerase of Streptomyces coelicolor A3(2); renaturation of a sigma subunit using GroEL. 135 Mar 15

1. A protein kinase type II was purified from calf thymus chromatin using ammonium sulphate fractionation, ion exchange chromatography on DEAE and phosphocellulose and affinity chromatography on phosvitin- and casein-sepharose columns. 2. The enzyme moves as a single band in non-denaturing gel electrophoresis at pH 8.3, which coincides with the enzyme activity assayed on gel slices. 3. Sodium dodecyl sulphate gel electrophoresis shows three separate polypeptide chains having M(r) of 40,000, 38,000 and 25,000, respectively. The native M(r) was about 130,000, as measured by HPLC on Superose 12 column, suggesting a subunit structure of alpha, alpha', beta 2 type. The enzyme incubated with [gamma 32P]ATP or [gamma 32P]GTP as phosphoryl donors undergoes autophosphorylation in the M(r) = 25,000 subunit. 4. The enzyme phosphorylates casein (Km = 7 microM) and phosvitin (Km = 5 microM) but not histones and was strongly deactivated by Zn2+ ions (I50 = 0.05 mM) and heparin (I50 = 0.1 micrograms/ml). 5. The enzyme seems to be the major phosphorylating system present in the 0.35 M NaCl chromatin extract of calf thymus. The RNA polymerase II from calf thymus and RNA polymerase from E. coli are both phosphorylated by protein kinase NII. The effect of phosphorylation, which causes a remarkable increase of DNA transcription rate, was studied in vitro and extensively discussed.
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PMID:Protein kinase NII from calf thymus chromatin. Isolation, characterization and some functional properties. 145 14

Earlier, our laboratory reported the cloning of a chromosomally encoded cholera toxin (CT)-like enterotoxin gene from Salmonella typhimurium Q1 into pBR322. Cell lysates from the plasmid clone pC1, containing a 4.8 kb EcoR1 DNA fragment from Salmonella, caused elongation of Chinese hamster ovary (CHO) cells and this biologic activity was neutralized by anti-CT. However, this cloned gene product did not elicit fluid secretion in the rabbit intestinal loop (RIL) model, because of poor expression. We report here, subcloning of a 4.8 kb EcoRI and a 2.7 kb HindIII/Eco Rl fragment into a high expression T7 RNA polymerase/promoter system. Cell lysates from these clones elicited fluid secretion in the RIL model, caused firm induration in rabbit skin and elongated CHO cells. These biological activities were neutralized by anti-CT. SDS-PAGE and subsequent fluorographic analysis of Escherichia coli, harboring recombinant plasmids in a T7 expression system, revealed the presence of two prominent 35S-labeled polypeptides of 25 and 12 kDa, which were immunoprecipitated with anti-CT. The enterotoxin appeared to be 125 kDa in size, based on chromatography on a P-300 column, had a pl of 6.6 to 6.8, and was heat-labile (60 degrees C/5 min). Unlike cloned CT and heat-labile enterotoxin (LT-l), which were localized in the periplasm, the Salmonella enterotoxin was cytoplasmic in nature.
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PMID:Expression and characterization of the cloned Salmonella typhimurium enterotoxin. 145 24

The Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been assembled from three components. An assembly pathway of these polypeptides, which specifies their interactions, has been determined. The TATA-binding protein, TBP, and the TFIIB-related BRF1 gene product BRF, together reconstitute the transcription factor activity and TFIIC-dependent DNA-binding activity of the B' component of TFIIIB. BRF alone weakly binds to a TFIIIC-tRNA gene complex; TBP greatly stabilizes this interaction. B" transcription factor activity is recovered with its previously identified 90 kd polypeptide from SDS-polyacrylamide gels. Incorporation of the 90 kd B" protein into the transcription complex requires TBP. The heparin-resistant TFIIIB-DNA complex retains all three of its constituent proteins, TBP, BRF, and B".
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PMID:The role of the TATA-binding protein in the assembly and function of the multisubunit yeast RNA polymerase III transcription factor, TFIIIB. 145 36


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