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)

Five cytotoxic Mannich bases (5-dimethylamino-1-substituted phenyl-1-penten-3-ones), three having antineoplastic activity, were evaluated for respiratory-inhibiting properties in rat liver mitochondria in the presence of four substrates: succinate, glutamate, 3-hydroxybutyrate, and palmitylcarnitine. Four compounds (Ib--Ie) showed significant inhibiting properties which, on occasion, were reversed partially by coenzyme Q10. Evaluation of the spectra of the mitochondrial cytochromes indicated that Ib--Ie blocked the electron transport chain prior to the sequence of cytochromes. Since inhibition occurred when different substrates were used, a common site of action for Ib--Ie is likely; competition of Ib--Ie with coenzyme Q10 probably occurs. Compounds Ia--Ie inhibited RNA polymerase from Swiss mouse kidney cells but were virtually bereft of activity versus RNA polymerase from L-1210 leukemia cells. Polarography of the Mannich bases and the related styryl ketones showed that antineoplastic activity was associated with higher half-wave potentials.
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PMID:Effect of antineoplastic and cytotoxic Mannich Bases derived from conjugated styryl ketones on mitochondrial respiration in rat liver cells. 71 88

Escherichia coli RNA polymerase can terminate transcription efficiently at rho-independent terminators in a purified transcription system in the absence of accessory factors. This process of "intrinsic termination" involves direct recognition of the terminator by the core RNA polymerase, and provides an important model system for the study of the molecular interactions involved in the switch between elongation and termination. We have analyzed the intrinsic termination efficiency (%T) of 13 rho-independent terminators, under a variety of in vitro reaction conditions. Although all of these sites share the general sequence features of typical rho-independent terminators, we find a wide range of %T (2% to 90%) for the different sites under our standard transcription conditions. While %T for a particular site is characteristic of that site, the efficiency can be altered considerably by the nature and concentration of salts in the reaction, by alteration of the concentrations of the nucleoside triphosphate substrates, or by transcription from supercoiled rather than linear templates. Surprisingly, different conditions can alter %T to a different extent for different terminators. For neutral salts such as potassium chloride or potassium glutamate, changes in the range from 0.1 to 1 M affect %T for different terminators in a distinct manner, depending on the terminator and the anion involved. At some sites, %T is greatly increased by Cl- concentrations up to 1 M, while at other sites %T is reduced or unaffected by these conditions. At some sites K+ concentrations up to 1 M give a modest increase in %T, while at other sites %T is slightly reduced under the same conditions. Thus the actual values of %T, as well as the order of terminator sites ranked according to %T, can be altered greatly according to the choice of reaction conditions. Reduction of the Mg2+ concentration below 1 mM has a dramatic and quite different effect, enhancing termination to approximately 100% for all terminators tested. Transcription of supercoiled DNA templates gives somewhat reduced %T as compared with linear DNA templates. However, the effect is no greater than twofold. Our results are not consistent with those expected for models in which %T is determined by the differential stability of DNA, RNA and hybrid duplex structures at the melted region in the transcription complex. Thus, the Cl anion does not affect the stability of nucleic acid duplexes even at 1 M concentrations, but can enhance termination tenfold. Also, the alterations of monovalent cation concentration that affect %T are not expected to have a differential effect on Tm for DNA, RNA and hybrid duplexes.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Parameters affecting transcription termination by Escherichia coli RNA polymerase. I. Analysis of 13 rho-independent terminators. 137 65

The rpoA341 (phs) mutation of Escherichia coli results in decreased expression of several positively regulated operons and has been mapped to within or very near the rpoA gene encoding the alpha subunit of RNA polymerase. We have shown that plasmid-directed synthesis of the wild-type alpha subunit can complement the defective phenotypes associated with this mutation consistent with its proposed location within rpoA. This mutation was mapped by marker rescue to within a 182bp region near the 3' end of rpoA and was subsequently transferred to a plasmid by recombination in vivo. DNA sequence analysis revealed that the RpoA341 phenotype was the result of the substitution of lysine 271 by glutamate within the alpha polypeptide. We discuss this result in relation to our current understanding of the functional organization of the alpha subunit.
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PMID:Escherichia coli rpoA mutation which impairs transcription of positively regulated systems. 177 61

The role of the co-transported cation in the coupling mechanism of the melibiose permease of Escherichia coli has been investigated by analysing its sugar-binding activity, facilitated diffusion reactions and energy-dependent transport reactions catalysed by the carrier functioning either as an H+, Na+ or Li(+)-sugar symporter. The results suggest that the coupling cation not only acts as an activator for sugar-binding on the carrier but also regulates the rate of dissociation of the co-substrates in the cytoplasm by controlling the stability of the ternary complex cation-sugar-carrier facing the cell interior. Furthermore, there is some evidence that the membrane potential enhances the rate of symport activity by increasing the rate of dissociation of the co-substrates from the carrier in the cellular compartment. Identification of the melibiose permease as a membrane protein of 39 kDa by using a T7 RNA polymerase/promoter expression system is described. Site-directed mutagenesis has been used to replace individual carrier histidine residues by arginine to probe the functional contribution of each of the seven histidine residues to the symport mechanism. Only substitution of arginine for His94 greatly interferes with the carrier function. It is finally shown that mutations affecting the glutamate residue in position 361 inactivate translocation of the co-substrates but not their recognition by the permease.
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PMID:The melibiose/Na+ symporter of Escherichia coli: kinetic and molecular properties. 197 Jun 46

A gene encoding a carrier protein for glutamate and aspartate was cloned into Escherichia coli K-12 strain BK9MDG by using the high-copy-number plasmid pBR322. The gene (designated gltP) is probably identical to a gene recently cloned from E. coli B (Y. Deguchi, I. Yamato, and Y. Anraku, J. Bacteriol. 171:1314-1319). A 1.6-kilobase DNA fragment containing gltP was subcloned into the expression plasmids pT7-5 and pT7-6, and its product was identified by a phage T7 RNA polymerase-T7 promoter coupled system (S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. USA 82:1074-1078) as a polypeptide with an apparent mass of 38 kilodaltons. A portion of the gltP polypeptide was associated with the cytoplasmic membrane. The nucleotide sequence of the 1.6-kilobase fragment was determined. It contained an open reading frame capable of encoding a highly hydrophobic polypeptide of 395 amino acids, containing four possible transmembrane segments. Uptake of glutamate and aspartate was increased 5.5- and 4.5-fold, respectively, in strains containing gltP plasmids. Glutamate uptake was insensitive to the concentration of Na+ and was inhibited by L-cysteate and beta-hydroxyaspartate. These results suggest that gltP is a structural gene for a carrier protein of the Na(+)-independent, binding-protein-independent glutamate-aspartate transport system.
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PMID:Cloning and sequencing of a gene encoding a glutamate and aspartate carrier of Escherichia coli K-12. 197 22

Osmoregulated transcription from the proU promoter of Escherichia coli has been successfully reconstituted from purified components in a simple in vitro system consisting of plasmid DNA template, RNA polymerase, and nucleotides in the absence of any other protein factor. proU transcription is stimulated by addition of high concentrations of potassium glutamate, the ionic compound accumulated in vivo during hyperosmotic stress. Transcription from the nonosmoregulated promoters beta la, lac, and pepN is inhibited under the same conditions, demonstrating the specificity of potassium glutamate as an inducer of proU transcription. proU transcription requires a circular DNA template, but stable alterations in the degree of supercoiling are unnecessary for this potassium glutamate-dependent signaling. These results agree well with previous data obtained in an S-30 coupled transcription/translation system and suggest that physiological changes in the ionic composition of the intracellular millieu can regulate gene expression.
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PMID:Osmotic control of proU transcription is mediated through direct action of potassium glutamate on the transcription complex. 197 30

Although protein-nucleic acid interactions exhibit dramatic dependences on both ion concentration and type in vitro, large variations in intracellular ion concentrations can occur in Escherichia coli and other organisms without apparent effects on gene expression in vivo. E. coli accumulates K+ and glutamate as cytoplasmic osmolytes. The cytoplasmic K+ concentration in E. coli varies from less than 0.2 to greater than 0.9 m as a function of external osmolarity; corresponding cytoplasmic glutamate concentrations range from less than 0.03 to greater than 0.25 m. Only low levels of chloride occur in the cytoplasm of E. coli at all osmotic conditions. Since most in vitro studies have been performed in chloride salts, whereas glutamate is the more relevant physiological anion, we have measured the effects of the substitution of potassium glutamate (KGlu) for KCl on the kinetics and equilibria of a variety of site-specific protein-DNA interactions in vitro. Both the interaction of E. coli RNA polymerase with two phage lambda promoters and the interactions of various restriction enzymes with their DNA cleavage sites are enhanced by this substitution. Using the abortive initiation assay, we find a greater than 30-fold increase in the second-order rate constant for open complex formation at the lambda PR promoter and a 10-fold increase at the lambda PR' promoter, when KGlu is substituted for KCl. Replacement of KCl by KGlu does not affect the strong salt dependences of these interactions; increasing either KCl or KGlu concentrations decreases both reaction rates and extents. Substitution of glutamate for chloride does, however, shift the range of salt concentrations over which these interactions are observable to higher K+ concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Replacement of potassium chloride by potassium glutamate dramatically enhances protein-DNA interactions in vitro. 288 98

Citrate synthase is a key enzyme of the Krebs tricarboxylic acid cycle and catalyzes the stereospecific synthesis of citrate from acetyl coenzyme A and oxalacetate. The amino acid sequence and three-dimensional structure of pig citrate synthase dimers are known, and regions of the enzyme involved in substrate binding and catalysis have been identified. A cloned complementary DNA sequence encoding pig citrate synthase has been isolated from a pig kidney lambda gt11 cDNA library after screening with a synthetic oligonucleotide probe. The complete nucleotide sequence of the 1.5-kilobase cDNA was determined. The coding region consists of 1395 base pairs and confirms the amino acid sequence of purified pig citrate synthase. The derived amino acid sequence of pig citrate synthase predicts the presence of a 27 amino acid N-terminal leader peptide whose sequence is consistent with the sequences of other mitochondrial signal peptides. A conserved amino acid sequence in the mitochondrial leader peptides of pig citrate synthase and yeast mitochondrial citrate synthase was identified. To express the pig citrate synthase cDNA in Escherichia coli, we employed the inducible T7 RNA polymerase/promoter double plasmid expression vectors pGP1-2 and pT7-7 [Tabor, S., & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074-1078]. The pig citrate synthase cDNA was modified to delete the N-terminal leader sequence; then by use of a synthetic oligonucleotide linker, the modified cDNA was cloned into pT7-7 immediately following the initiator Met. A glutamate-requiring (citrate synthase deficient), recA- E. coli mutant, DEK15, was transformed with pGP1-2 and then pT7-7PCS. pT7-7PCS complemented the E. coli gltA mutation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation, nucleotide sequence, and expression of a cDNA encoding pig citrate synthase. 304 87

Effects of changes in intracellular ion concentrations on the interactions of Escherichia coli lac repressor with lac operator mutants and on the interactions of RNA polymerase with various promoters have been investigated in vivo. The intracellular ionic environment was reproducibly varied by changing the osmolality of the 4-morpholinepropanesulfonic acid minimal growth medium. As the osmolality of the growth medium is varied from 0.1 to 1.1 osmolal, the total intracellular concentration of K+ increases linearly from 0.23 +/- 0.03 to 0.93 +/- 0.05 molal and the total intracellular concentration of glutamate increases linearly from 0.03 +/- 0.01 to 0.26 +/- 0.02 molal. The sum of the changes in the total concentrations of these two ions appears sufficient to compensate for a given change in external osmolality, indicating that K+ and glutamate are the primary ionic osmolytes under these conditions and that these ions are free in the cytoplasm. In support of this, in vivo 39K NMR experiments as a function of external osmolality indicate that changes in the total cytoplasmic K+ concentration correspond to changes in the free cytoplasmic K+ concentration. Extents of interaction of lac repressor and RNA polymerase with their specific DNA sites were monitored by measuring the amounts of beta-galactosidase produced under the control of these sites. For both lac repressor and RNA polymerase, it was found that formation of functional protein-DNA complexes in vivo is only weakly (if at all) dependent on intracellular ion concentration. These results contrast strongly with those obtained on these systems in vitro, which showed that both the equilibria and kinetics of binding are extremely salt-dependent. We discuss several possible mechanisms by which E. coli may compensate for the potentially disruptive effects of these large changes in the intracellular ionic environment.
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PMID:Variability of the intracellular ionic environment of Escherichia coli. Differences between in vitro and in vivo effects of ion concentrations on protein-DNA interactions and gene expression. 310 49

A novel procedure of operational ease and reproducibility for the partial purification of DNA-dependent RNA polymerase [EC 2.7.7.6] from Escherichia coli is reported. It utilizes liquid phase partitions with polyethylene glycol and Dextran, ammonium sulfate fractionations and chromatography on a QAE-Sephadex A-50 column. A copurified protein in the partially purified preparation was isolated and identifed as glutamine synthetase [L-glutamate : ammonia ligase (ADP) EC 6.3.1.2].
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PMID:Glutamine synthetase in preparations of RNA polymerase of Escherichia coli: a novel purification procedure. 612 39


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