Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The amatoxins, highly toxic components of Amanita mushrooms, strongly inhibit the DNA-dependent RNA polymerase II (or B) in eukaryotic cell nuclei. For optimal binding to the enzyme a gamma-hydroxyisoleucine side chain in the 3-position is important as in gamma-amanitin (compound 1), where the OH-group is bound in the [S]-configuration. Amanullin, a non-toxic component, having an oxygen-free isoleucine side chain no. 3, exhibits an inhibitory effect on RNA polymerase II about two orders of magnitude smaller than that of gamma-amanitin. An equal, relatively weak, inhibitory effect has previously been found with the synthetically obtained Ile3-analog 7. In the present paper the synthesis of an analog (2) bearing a gamma-hydroxyl group in the isoleucine side chain is described. The compound was found to have about the same inhibitory effect on RNA polymerase II from Drosophila embryos as amanullin and the Ile3-analog 7. Structure analysis by X-ray diffraction revealed that the hydroxyl group at the -carbon atom of side chain-3 has the [R]-configuration, the new analog thus being -deoxo[( )-hydroxy-[Ile3]-amaninamide. It follows that the [S]-configuration of this chiral center is a prerequisite to maximal toxicity. Crystallographic data demonstrating great similarity between the peptide backbones of the new analog and those of natural amatoxins are given.
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PMID:Structure-toxicity relationships in the amatoxin series. Synthesis of S-deoxy[gamma(R)-hydroxy-Ile3]-amaninamide, its crystal and molecular structure and inhibitory efficiency. 259 60

The ilvC gene encodes acetohydroxy acid isomeroreductase (EC 1.1.1.89), the second enzyme in the parallel isoleucine-valine biosynthetic pathway. Expression of the ilvC gene is induced by acetohydroxy acid isomeroreductase substrates, acetohydroxybutyrate or acetolactate. This substrate induction is mediated by a positive activator encoded by an adjacent gene, ilvY. The ilvY and ilvC genes are transcribed in opposite directions from promoters that are overlapping. In this paper we characterize the in vitro DNA binding properties of the ilvY-encoded activator protein. The ilvY product binds to two adjacent operator sites located in the divergent-overlapping ilvY and ilvC promoter region. One of these operators, designated O1 contains regions of dyad symmetry centered at position +17 relative to the ilvY transcriptional start site, and the second site, designated O2, contains an homologous inverted repeat sequence centered about the -35 region of the ilvC promoter. Binding of the ilvY product at the O1 and O2 operator sites is co-operative and this ilvY protein-DNA complex in the presence of acetohydroxy acid isomeroreductase substrate is a prerequisite for RNA polymerase binding to the ilvC promoter as detected by DNase I protection experiments. Additionally, chromosomal galK transcriptional fusion assays were performed to characterize the regulation of the ilvY and ilvC promoters in vivo. Transcription of the ilvC gene is maintained at a basal level of activity which is elevated as much as 15-fold in the presence of ilvY product and acetohydroxybutyrate. The ilvY product represses ilvY transcription in a manner that does not appear to be dependent on acetohydroxy acid isomeroreductase substrate. We discuss models in which activation of ilvC transcription results from a direct interaction of ilvY protein with RNA polymerase or an ilvY-mediated alteration of the DNA conformation of the ilvC -35 promoter region. Additionally, we discuss the role of acetohydroxybutyrate and acetolactate in ilvY transcriptional regulation.
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PMID:Transcriptional activation at adjacent operators in the divergent-overlapping ilvY and ilvC promoters of Escherichia coli. 306 77

The nucleotide sequence (25,320 base-pairs) of a part of the large single-copy region of chloroplast DNA from the liverwort Marchantia polymorpha was determined. This region encodes putative genes for four tRNAs, isoleucine tRNA(CAU), arginine tRNA(CCG), proline tRNA(UGG) and tryptophan tRNA(CCA); eight photosynthetic polypeptides, the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL), 51,000 Mr photosystem II chlorophyll alpha apoprotein (psbB), apocytochrome b-559 polypeptides (psbE and psbF), 10,000 Mr phosphoprotein (psbH), cytochrome f preprotein (petA), cytochrome b6 polypeptide (petB), and cytochrome b6/f complex subunit 4 polypeptide (petD); 13 ribosomal proteins (L2, L14, L16, L20, L22, L23, L33, S3, S8, S11, S12, S18 and S19); initiation factor 1 (infA); ribosome-associating polypeptide (secX); and alpha subunit of RNA polymerase (rpoA). Functionally related genes were located in several clusters in this region of the genome. There were two ribosomal protein gene clusters: rpl23-rpl2-rps19-rpl22-rps3-rpl16-+ ++rpl14-rps8-infA-secX-rps11-rpoA, with a gene arrangement similar to that of the Escherichia coli S10-spc-alpha operons, and the rps12'-rpl20-rps18-rpl33 cluster. There were gene clusters encoding photosynthesis components such as the psbB-psbH-petB-petD and the psbE-psbF clusters. Thirteen open reading frames, ranging in length from 31 to 434 amino acid residues, remain to be identified.
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PMID:Structure and organization of Marchantia polymorpha chloroplast genome. III. Gene organization of the large single copy region from rbcL to trnI(CAU). 319 36

The growth of MCF-7 cells was arrested by 24 h of isoleucine deprivation. Following replenishment of the medium, the incorporation of uridine and thymidine into trichloroacetic acid-precipitable material began to increase slowly and gradually rose to the level of cycling cells. The addition of 5 X 10(-9) M estradiol to growth-arrested cells dramatically shortened the time of onset of macromolecular synthesis and increased the overall amount of precursor incorporation 2- to 4-fold over the level obtained by arrested control cells. The increase in uridine incorporation preceded the increase in thymidine incorporation by 6 h. Inhibition of protein synthesis with cycloheximide blocked the recovery of macromolecular synthesis in both control and estrogen-treated cells. Actinomycin D was ineffective in blocking the estrogen-stimulated recovery of macromolecular synthesis at concentrations known to inhibit pre-rRNA synthesis (10(-8) M). At higher concentrations, uridine and thymidine incorporation were inhibited in a dose-dependent manner. Inhibition of RNA polymerase II activity with alpha-amanitin similarly blocked both the recovery of the cells from isoleucine starvation and the potentiation of this by estradiol. Dihydrofolate reductase and thymidine kinase activities are both stimulated by estradiol in MCF-7 cells. In cycling cells, estrogen stimulates a 2-fold increase in their messenger RNAs (mRNAs) within 24 h. The level of dihydrofolate reductase mRNA is unaffected by isoleucine starvation, and estrogen caused no change in dihydrofolate reductase mRNA levels over a 24-h period following reversal of growth arrest. Similar results were observed for the 600-nucleotide pS2 mRNA that has been identified as an estrogen-induced RNA in MCF-7 cells. In contrast, thymidine kinase mRNA was found to be increased by estrogen at 24 h, but not at 12 h, following reversal of growth arrest. This increase correlates with increases in thymidine, but not uridine incorporation. These data indicate that the estrogen-stimulated increase in thymidine incorporation following release from growth arrest is dependent on new RNA synthesis. However, the hormone did not increase the levels of three estrogen-regulated mRNAs coordinately with the increases observed in uridine incorporation.
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PMID:Relationship between the expression of estrogen-regulated genes and estrogen-stimulated proliferation of MCF-7 mammary tumor cells. 398 99

The production by Neurospora of the enzymes of isoleucine and valine synthesis in response to specific end product-derived signals depends upon the presence of an effective leu-3 regulatory product and its effector alpha-isopropylmalate (alpha-IPM). In leu-3(+) strains, threonine deaminase production is repressed as a function of available isoleucine, acetohydroxy acid synthetase as a function of valine, and the isomeroreductase and dihydroxy acid dehydratase as a function of isoleucine and leucine. In the absence of an effective leu-3 regulatory product, alpha-isopropylmalate, or both, the production of isoleucine and valine biosynthetic enzymes is fixed at or near fully repressed levels even under conditions of severe end product limitation. Thus, in addition to its involvement in the regulation of expression of the three structural genes of leucine synthesis, the leu-3 alpha-IPM regulatory product is necessary for full expression of at least four genes specifying the structure of the enzymes of isoleucine and valine synthesis. It is suggested that the leu-3 alpha-IPM regulatory element may facilitate transcription of the genetically dispersed cistrons either by imposing specificity on ribonucleic acid polymerase for structurally similar promoters adjacent to each of the cistrons or by "opening" promoters after interaction with nearly identical stretches of deoxyribonucleic acid near each of the structural genes.
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PMID:Role of the leu-3 cistron in the regulation of the synthesis of isoleucine and valine biosynthetic enzymes of Neurospora. 482 4

Growth of phase alpha 3a on stationary phase Vibrio cultures requires micro-aerophilic conditions and is inhibited by aeration. Since pre-conditioning of the bacteria by allowing them to stand for 24 h after shaking for 3 d is an important aspect of the stationary phase phage growth system, various physiological and morphological characteristics of the stationary phase cells during the transition from shaking to standing were investigated. Shaken stationary phase cells were less viable and more sensitive to ultraviolet irradiation and heat than standing stationary phase cells. During pre-conditioning the small, non-flagellated cells present in shaken stationary phase cultures underwent morphological changes and became large, flagellated rods which resembled exponential phase cells. The transition of stationary phase cells from shaking to standing was associated with a marked increase in total RNA synthesis but a rapid and large decrease in total protein synthesis. Intracellular concentrations of ATP in shaken stationary phase cells were 53% lower than those in standing stationary phase cells. Studies on leucine uptake indicated that its transport was inhibited by isoleucine and that the major part (90%) of the total leucine uptake was due to a shared system for uptake of both amino acids. Shaken stationary phase cells transported less leucine than standing stationary phase cells. Inhibition of phage growth in aerated stationary phase cultures was not due to the prevention of phase absorption by shaking. It is suggested that the observed differences between shaken and standing stationary phase cells could be due to aeration affecting the template specificity of the Vibrio RNA polymerase.
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PMID:Physiological and morphological characteristics of stationary phase vibrio cells able to support phase growth. 616 43

The inhibition constants (Ki) of DNA-dependent RNA polymerase B (or II) from calf thymus were measured for eight synthetically obtained bicyclic amanitin-like thioethers, two R-sulfoxides, and two S-sulfoxides. These Ki values were compared with those of alpha-amanitin, its 6'-O-methylether Ia (an R-sulfoxide), the S-sulfoxide, the sulfone, the S-deoxo derivative (Id) of Ia, and several previously described amatoxins. The necessity of a beta-methyl side chain in position 3 and a hydroxy group in proline-2 was confirmed. Additionally, the presence of an isoleucine side chain in position 6 and the absence of a side chain in position 5 was recognized as important for binding to the enzyme. In the three sulfoxide samples examined, the R-diastereomer was found to be a stronger inhibitor than the S-form. The contribution of every structural element to biological activity has been discussed.
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PMID:The effect of the chemical nature of the side chains of amatoxins in the inhibition of eukaryotic RNA polymerase B. 726 84

Inophyllums are novel non-nucleoside inhibitors of human immunodeficiency virus (HIV) type 1 reverse transcriptase identified through an enzyme screening program and isolated from the plant Calophyllum inophyllum. The kinetics of reverse transcriptase inhibition by inophyllum B were characterized using recombinant purified enzyme, a heteropolymeric RNA template, and a scintillation proximity assay. Preincubation of inhibitor with the enzyme-template-primer complex for 11 min was required for maximal inhibition of reverse transcriptase to occur, suggesting that inophyllum B had a slow on-rate and that template-primer must bind to reverse transcriptase prior to inhibitor binding. Inhibition of reverse transcriptase by inophyllums was shown to be reversible. When thymidine triphosphate was the variable substrate, inophyllum B inhibited reverse transcriptase noncompetitively with a Ki of 42 nM. Enzyme inhibition with respect to template-primer was uncompetitive with a Ki of 26 nM. Reverse transcriptase enzymes containing point mutations in which tyrosine 181 was changed to either cysteine or isoleucine exhibited marginal resistance to inophyllums but were resistant to (+)-(5S)-4,5,6,7-tetrahydro-9-chloro-5-methyl-6- (3-methyl-2-butenyl)-imidazo[4,5,1-j,k][1,4]benzodiazepin-2-(1H)-t hione (TIBO R82913). A mutant enzyme in which tyrosine 188 was changed to leucine was cross-resistant to both inophyllum B and TIBO R82913, as was HIV type 2 reverse transcriptase. These studies suggest that inophyllum B and TIBO R82913 bind to distinct but overlapping sites. Inhibition of avian myeloblastosis virus reverse transcriptase and Moloney murine leukemia virus reverse transcriptase by inophyllum B was detectible, suggesting that these inhibitors may be more promiscuous than other previously described non-nucleoside inhibitors. Inophyllums were active against HIV type 1 in cell culture with IC50 values of approximately 1.5 microM. These studies imply that the inophyllums have a novel mechanism of interaction with reverse transcriptase and as such could conceivably play a role in combination therapy.
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PMID:Kinetic and mutational analysis of human immunodeficiency virus type 1 reverse transcriptase inhibition by inophyllums, a novel class of non-nucleoside inhibitors. 750

The ability of bovine mitochondrial tRNA(Met) with the anticodon f5CAU (where f5C is 5-formylcytidine) to decode AUG and AUA codons was examined in a codon-dependent ribosomal binding assay. The AUG codon stimulated the binding of Met-tRNA(Met) to mitochondrial ribosomes in the presence of EF-Tu/TSmt. In contrast, the AUA codon did not promote the binding to mitochondrial Met-tRNA to the ribosome. To investigate the translation of the AUG and AUA codons more fully, an in vitro translation system from bovine liver mitochondria was developed. The activity of this system was greatly enhanced by the addition of 1 mM spermine and reached about half the activity observed with a comparable translational system from E coli. Two types of mRNA containing either AUG or AUA codons were synthesized using T7 RNA polymerase to transcribe their chemically synthesized genes. In the E coli system, the AUG-containing mRNA was translated as Met and the AUA-containing mRNA was translated as Ile. The AUG-containing mRNA but not the AUA-containing mRNA was translated as Met by the mitochondrial translational system. The process by which the AUA codon is translated as Met in the mitochondrial system remains to be clarified.
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PMID:The ability of bovine mitochondrial transfer RNAMet to decode AUG and AUA codons. 759 68

The (-) enantiomers of 2',3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC] and 2',3'-dideoxy-3'-thiacytidine [(-)-BCH-189] were recently shown to inhibit selectively human immunodeficiency viruses (HIV) and hepatitis B virus in vitro. In the current study, the potential for HIV type 1 (HIV-1) resistance to these compounds was evaluated by serial passage of the virus in human peripheral blood mononuclear cells and MT-2 cells in the presence of increasing drug concentrations. Highly drug-resistant HIV-1 variants dominated the replicating virus population after two or more cycles of infection. The resistant variants were cross-resistant to (-)-FTC, (-)-BCH-189, and their (+) congeners but remained susceptible to 2',3'-dideoxycytidine, 3'-azido-3'-deoxythymidine, 3'-fluoro-3'-deoxythymidine, 2',3'-dideoxyinosine, phosphonoformate, the TIBO compound R82150, and the bis(heteroaryl)piperazine derivative U-87201E. Reverse transcriptase derived from drug-resistant viral particles was 15- to 50-fold less susceptible to the 5'-triphosphates of FTC and BCH-189 compared with enzyme from parental drug-susceptible virus. DNA sequence analysis of the reverse transcriptase gene amplified from resistant viruses consistently identified mutations at codon 184 from Met (ATG) to Val (GTG or GTA) or Ile (ATA). Sequence analysis of amplified reverse transcriptase from a patient who had received (-)-BCH-189 therapy for 4 months demonstrated a mixture of the Met-184-to-Val (GTG) mutation and the parental genotype, indicating that the Met-184 mutation can occur in vivo.
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PMID:Characterization of human immunodeficiency viruses resistant to oxathiolane-cytosine nucleosides. 768 16


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