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

---

Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of DNA gyrase and quinolone drugs on in vitro transcription of a template containing a preferred gyrase cleavage site have been investigated. We have found that gyrase-quinolone complexes with DNA lead to blocking of transcription by Escherichia coli and bacteriophage T7 RNA polymerases. Either gyrase or quinolone alone has no effect on transcription. With DNA gyrase containing a point mutation in the gyrase A protein, known to confer quinolone resistance, blocking was found to occur only at much higher concentrations of the drug. Other agents that inhibit gyrase-catalysed supercoiling (novobiocin and 5'-adenylyl-beta,gamma-imidodiphosphate) do not arrest transcription in the presence of gyrase. Mapping of the transcription termination sites in the presence of gyrase and quinolones shows that blocking occurs about 10 to 20 base-pairs upstream of the gyrase cleavage site. Analysis of transcription in the absence of drug suggests that RNA polymerase does not displace gyrase from the template. These results are discussed in the light of models for the bactericidal effects of quinolone drugs.
...
PMID:The complex of DNA gyrase and quinolone drugs with DNA forms a barrier to transcription by RNA polymerase. 793 95

Phage P4 DNA is replicated in cell-free extracts of Escherichia coli in the presence of partially purified P4 alpha protein [Krevolin and Calendar (1985), J. Mol. Biol. 182, 507-517]. Using a modified in vitro replication assay, we have further characterized this process. Analysis by agarose gel electrophoresis and autoradiography of in vitro replicated molecules demonstrates that the system yields supercoiled monomeric DNA as the main product. Electron microscopic analysis of in vitro generated intermediates indicates that DNA synthesis initiates in vitro mainly at ori, the origin of replication used in vivo. Replication proceeds from this origin bidirectionally, resulting in theta-type molecules. In contrast to the in vivo situation, no extensive single-stranded regions were found in these intermediates. The initiation proteins of the host, DnaB and DnaG, and the chaperones DnaJ and DnaK are not required for P4 replication, because polyclonal antibodies against those polypeptides do not inhibit the process. The reaction is inhibited by antibodies against the SSB protein, and by ara-CTP, a specific inhibitor of DNA polymerase III holoenzyme. Consistent with previous reports, P4 in vitro replication is independent of transcription by host RNA polymerase. Novobiocin, a DNA gyrase inhibitor, strongly inhibits P4 DNA synthesis, indicating that form I DNA is the required substrate.
...
PMID:Phage P4 DNA replication in vitro. 802 13

Most DNA topoisomerase II (topo II) in cell-free extracts of 0-2-h old Drosophila embryos appears to be nonnuclear and remains in the supernatant after low-speed centrifugation (10,000 g). Virtually all of this apparently soluble topo II is particulate with a sedimentation coefficient of 67 S. Similar topo II-containing particles were detected in Drosophila Kc tissue culture cells, 16-19-h old embryos and extracts of progesterone-matured oocytes from Xenopus. Drosophila topo II-containing particles were insensitive to EDTA, Triton X-100 and DNase I, but could be disrupted by incubation with 0.3 M NaCl or RNase A. After either disruptive treatment, topo II sedimented at 9 S. topo II-containing particles were also sensitive to micrococcal nuclease. Results of chemical cross-linking corroborated those obtained by centrifugation. Immunoblot analyses demonstrated that topo II-containing particles lacked significant amounts of lamin, nuclear pore complex protein gp210, proliferating cell nuclear antigen, RNA polymerase II subunits, histones, coilin, and nucleolin. Northern blot analyses demonstrated that topo II-containing particles lacked U RNA. Thus, current data support the notion that nonnuclear Drosophila topo II-containing particles are composed largely of topo II and an unknown RNA molecule(s).
...
PMID:An RNase-sensitive particle containing Drosophila melanogaster DNA topoisomerase II. 808 68

DNA sequence analysis of Escherichia coli parC and parE, encoding the subunits of topoisomerase IV (Topo IV) (Kato, J.-I., Suzuki, H., and Ikeda, H. (1992) J. Biol. Chem. 267, 25676-25684), showed that ParC was 22 amino acids longer on the N terminus and ParE was 29 amino acids longer on the C terminus than reported previously. E. coli strains bearing bacteriophage T7 RNA polymerase-based expression plasmids carrying both intact and truncated parC and parE were used to overproduce the ParC and ParE proteins. Full-length ParC and ParE were required to reconstitute Topo IV activity, whereas the truncated ParC and ParE were inactive. Topo IV activity was supported only by ATP or dATP. The [ATP]1/2 for DNA relaxation was 0.45 mM, almost 25-fold higher than the [ATP]1/2 for decatenation of kinetoplast DNA. Topo IV activity was inhibited by the quinolone and coumarin antibiotics, although the concentrations required for 50% inhibition of activity were 3-30-fold higher than those required to inhibit DNA gyrase. The norfloxacin-induced DNA cleavage patterns of Topo IV and DNA gyrase were distinct but overlapping. The native forms of ParC and ParE were a dimer and a monomer, respectively; whereas the active form of Topo IV was a heterotetramer, ParC2ParE2. The inactivity of the truncated forms of ParC and ParE could be attributed to their failure to form the heterotetramer.
...
PMID:Escherichia coli topoisomerase IV. Purification, characterization, subunit structure, and subunit interactions. 822

Supercoiled plasmid DNAs with negative superhelicity several times higher than normal have been isolated from Escherichia coli topA mutants. The formation of these hypernegatively supercoiled plasmid DNAs is apparently induced by transcription. We show that hypernegatively supercoiled plasmid DNAs isolated from topA mutants contain R-loop(s). To study the mechanism of formation of hypernegatively supercoiled plasmid DNA, we have been able to reproduce hypernegatively supercoiled DNA in vitro using purified RNA polymerase and DNA gyrase. The formation of hypernegatively supercoiled plasmid DNA template in vitro is shown to require transcription elongation and is tightly linked to R-loop formation. We propose that one of the roles of topoisomerase I is to suppress R-loop formation during transcription elongation.
...
PMID:Hypernegative supercoiling of the DNA template during transcription elongation in vitro. 829 58

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.
...
PMID:DNA topology affects transcriptional regulation of the pertussis toxin gene of Bordetella pertussis in Escherichia coli and in vitro. 839 6

The primary target for the quinolone group of antibacterial agents is DNA gyrase. One model for the interaction of quinolone drugs with gyrase and DNA suggests that the drugs bind to the single-stranded regions revealed following DNA cleavage by the enzyme. We have tested this hypothesis by using mutants which have the active-site tyrosine in the gyrase A subunit altered to phenylalanine or serine. We have found that proteins bearing these mutations are still able to bind drug, suggesting that DNA cleavage is not a prerequisite for drug binding. We have also found that the blocking of transcription by RNA polymerase in vitro by the gyrase-quinolone complex on DNA does not occur when the active-site tyrosine is mutated to serine; i.e., polymerase blocking requires DNA cleavage.
...
PMID:DNA cleavage is not required for the binding of quinolone drugs to the DNA gyrase-DNA complex. 865 15

The primary theme emerging from molecular genetic work conducted with Mycobacterium tuberculosis and several other mycobacterial species is that resistance is commonly associated with simple nucleotide alterations in target chromosomal genes rather than with acquisition of new genetic elements encoding antibiotic-altering enzymes. Mutations in an 81-bp region of the gene (rpoB) encoding the beta subunit of RNA polymerase account for rifampin resistance in 96% of M. tuberculosis and many Mycobacterium leprae isolates. Streptomycin resistance in about one-half of M. tuberculosis isolates is associated with missense mutations in the rpsL gene coding for ribosomal protein S12 or nucleotide substitutions in the 16S rRNA gene (rrs). Mutations in the katG gene resulting in catalase-peroxidase amino acid alterations nad nucleotide substitutions in the presumed regulatory region of the inhA locus are repeatedly associated with isoniazid-resistant M. tuberculosis isolates. A majority of fluoroquinolone-resistant M. tuberculosis isolates have amino acid substitutions in a region of the DNA gyrase A subunit homologous to a conserved fluoroquinolone resistance-determining region. Multidrug-resistant isolates of M. tuberculosis arise as a consequence of sequential accumulation of mutations conferring resistance to single therapeutic agents. Molecular strategies show considerable promise for rapid detection of mutations associated with antimicrobial resistance. These approaches are now amenable to utilization in an appropriately equipped clinical microbiology laboratory.
...
PMID:Antimicrobial agent resistance in mycobacteria: molecular genetic insights. 866 67

Salmonella typhimurium and Escherichia coli cells have two different class I ribonucleotide reductases encoded by the nrdEF and nrdAB operons. Despite the presence of one additional ribonucleotide reductase, the nrdAB-encoded enzyme is essential to the aerobic growth of the cell because nrdAB-defective mutants of both species are not viable in the presence of oxygen. Several factors controlling nrdAB gene transcription have been analysed intensively. Nothing is known about the expression of the nrdEF genes. To study this subject, and after cloning of E. coli nrdEF genes and sequencing of their 5' ends, the promoter of this operon has been identified by primer extension in both bacterial species. The +1 position was 691 bp and 692 bp upstream of the translational start points of the nrdE genes of S. typhimurium and E. coli, respectively. Downstream of the +1 position, and before the nrdE gene, two open reading frames (ORFs) of 81 and 136 amino acid residues are present in both bacteria. The synthesis of a polypeptide with a molecular mass of 9 kDa, corresponding to the first of these two ORFs, was observed by using the T7 RNA polymerase expression system. Comparison of the amino acid predicted sequence of this ORF reveals a significant similarity with glutaredoxin proteins. Competitive, reverse-transcription polymerase chain reaction experiments indicate that transcription from the nrdEF promoter normally takes place in wild-type cells. nrdEF transcription is increased by hydroxyurea, which inhibits class I ribonucleotide reductase activity, in both RecA+ and RecA- cells. nrdA(ts) mutants show a higher level of nrdEF transcription than wild-type cells at either the permissive or the restrictive temperature. nrdEF expression was unaffected by changes in DNA supercoiling whether caused by the introduction of either topA::Tn10 and hns::Tn10 mutations or by the inhibition of DNA gyrase with the antibiotic novobiocin. In contrast to the nrdAB genes, the nrdEF operon is not essential to the cells because nrdEF-defective mutants are viable under both aerobic and anaerobic conditions.
...
PMID:Promoter identification and expression analysis of Salmonella typhimurium and Escherichia coli nrdEF operons encoding one of two class I ribonucleotide reductases present in both bacteria. 882 Jun 48

Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.
...
PMID:Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases. 912 28


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---