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)

Adenosine 2',3'-riboepoxide 5'-triphosphate (epoxyATP) has been found to be a suicidal inactivator of DNA polymerase I from Escherichia coli by the following criteria. Inactivation is complete, is first order in enzyme activity, and shows saturation kinetics with an apparent KD of 30 +/- 10 micron for epoxy ATP. This KD is comparable to the KM of the substrate dATP. The t1/2 for inactivation is 1.3 min. Inactivation requires Mg2+ and the complementary template. The enzyme is protected by dATP but not by an excess of template. Gel filtration of the reaction mixture after inactivation with [3H]epoxy ATP results in the comigration of E. coli DNA polymerase I, the tritium-labeled inactivator, and the DNA template. The stoichiometry of binding approaches 1 mol of [3H]epoxy nucleotide per mol of inactivated enzyme. These results are consistent with the hypothesis that epoxy ATP initially serves as a substrate for the polymerase reaction, elongating the DNA chain by a nucleotidyl unit, and subsequently alkylates an essential base at the primer terminus binding site of the enzyme. Epoxy ATP also inactivates human and viral DNA polymerases but not E. coli RNA polymerase or rabbit muscle pyruvate kinase. Hence epoxy ATP may be a specific suicide reagent for DNA polymerases.
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PMID:Apparent suicidal inactivation of DNA polymerase by adenosine 2',3'-riboepoxide 5'-triphosphate. 34 91

A method for the rapid and quantitative analysis of 5'-terminal oligonucleotides of RNAs made in vitro is described. The method involves synthesis of RNA in the presence of [gamma-32P]ATP or GTP, isolation of the RNA, and digestion with T1 or pancreatic ribonucleases to release labeled 5'-triphosphate termanated oligonucleotides. The oligonucleotides are then subjected to chromatography on a polyethyleniminecellulose thin-layer system using 2 M LiCl, 0.01 M EDTA (pH 6.5) in the first dimension and 1.5 M LiCl, 1.8 M formic acid, 0.005 M EDTA (pH 2.0) in the second. RNAs made with E. coli RNA polymerase and lambdacb2, T7, T4, and adenovirus 2 DNA yield characteristic fingerprint patterns. The utility of this method in studying selectivity of in vitro RNA chain initiation is discussed.
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PMID:Selectivity of RNA chain initiation in vitro. 1. Analysis of RNA initiations by two-dimensional thin-layer chromatography of 5'-triphosphate-labeled oligonucleotides. 35 90

Purine riboside (nebularine, 9-beta-ribofuranosylpurine) is a naturally occurring base analog which closely resembles adenosine. It inhibits carcinogenic growth. Purine riboside strongly inhibits RNA and DNA synthesis in different cancer ascites cells. Gel electrophoretic analysis of RNA synthesis in vivo in the presence of purine riboside shows the ribosomal components to be inhibited the most. A method for assaying purine riboside or its phosphates intracellularly has been devised, and by using this it has been shown that purine riboside is extensively phosphorylated in the cells. The triphosphate derivative of purine riboside has been isolated and tested in the Escherichia coli RNA polymerase assay. It appears not to be incorporated into this type of RNA and to competitively inhibit this reaction with regard to ATP.
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PMID:Effects of purine riboside on nucleic acid synthesis in ascites cells. 35 98

A new assay yielding mechanistic information on the initiation reaction of Escherichia coli RNA polymerase has been developed. It was found to be useful in characterizing the promoters of bacteriophage DNA templates. The binding of the first two triphosphates in an RNA sequence was determined to be equilibrium ordered with ATP binding first followed by UTP on the lambda promoters PL. and PR. The products resulting from phosphodiester bond formation, pppApU and PPi, dissociated rapidly in the absence of the other triphosphates required for RNA synthesis. The resulting steady state conversion of ATP and UTP into pppApU was the basis for the new assay. The rate-limiting step in the initiation reaction was not precisely determined, but it was argued not to be entirely the release of product. The Zn2+ chelator, 1,10-phenanthroline, was partially characterized and found to be an uncompetitive inhibitor of ATP in the reaction (Ki = 100 micrometer). The unique advantage of this steady state assay is that several steps in the RNA initiation process are amplified kinetically and thus can be examined separately with techniques applicable to any other two-substrate, two-product enzyme reaction.
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PMID:A steady state assay for the RNA polymerase initiation reaction. 36 12

The mode of action of the antibiotic pseudomonic acid has been studied in Escherichia coli. Pseudomonic acid strongly inhibits protein and RNA synthesis in vivo. The antibiotic had no effect on highly purified DNA-dependent RNA polymerase and showed only a weak inhibitory effect on a poly(U)-directed polyphenylalanine-forming ribosomal preparation. Chloramphenicol reversed inhibition of RNA synthesis in vivo. Pseudomonic acid had little effect on RNA synthesis in a regulatory mutant, E. coli B AS19 RC(rel), whereas protein synthesis was strongly inhibited. In pseudomonic acid-treated cells, increased concentrations of ppGpp, pppGpp and ATP were observed, but the GTP pool size decreased, suggesting that inhibition of RNA synthesis is a consequence of the stringent control mechanism imposed by pseudomonic acid-induced deprivation of an amino acid. Of the 20 common amino acids, only isoleucine reversed the inhibitory effect in vivo. The antibiotic was found to be a powerful inhibitor of isoleucyl-tRNA synthetase both in vivo and in vitro. Of seven other tRNA synthetases assayed, only a weak inhibitory effect on phenylalanyl-tRNA synthetase was observed; this presumably accounted for the weak effect on polyphenylalanine formation in a ribosomal preparation. Pseudomonic acid also significantly de-repressed threonine deaminase and transaminase B activity, but not dihydroxyacid dehydratase (isoleucine-biosynthetic enzymes) by decreasing the supply of aminoacylated tRNA(Ile). Pseudomonic acid is the second naturally occurring inhibitor of bacterial isoleucyl-tRNA synthetase to be discovered, furanomycin being the first.
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PMID:Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Escherichia coli by pseudomonic acid. 36 75

Transcription was determined in liver chromatin from rats fed for 6 days, an optimal (20%) or suboptimal (3%) amount of high-quality protein. Transcription by Escherichia coli RNA polymerase (EC 2.7.7.6) was lower after prolonged incubation with chromatin from rats fed 3% as compared with 20% protein. Differences were detected in the transcripts of the two types of chromatin after analysis by sucrose density gradient centrifugation. But no measurable differences were found in the melting profiles at low ionic strength of the two chromatin preparations. Transcription per milligram chromatin DNA was 25-fold higher using E. coli RNA polymerase instead of rat liver RNA polymerase II. The use of UTP as radioactive precursor in the absence of ATP, GTP and CTP resulted in a low labelling of RNA. One [lambda32P]UTP nucleotide was incorporated/8 UMP nucleotides. The product obtained was sensitive to ribonuclease treatment. In the presence of ATP, GTP and CTP [lambda-32P]UTP nucleotide incorporation was reduced and that of UMP nucleotide was increased giving a ratio of 1:188.
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PMID:Transcription of rat liver chromatin by Escherichia coli RNA polymerase: template properties after protein restriction. 36 67

E. coli DNA dependent RNA polymerase was modified by diethylpyrocarbonate. Optical and kinetic properties of the reaction were studied. More than 90% of RNA polymerase activity is inhibited by introduction of 9--11 ethoxyformyl groups per enzyme molecule without loss of its ability to bind DNA template. Furthermore the modified enzyme is able to form tight complexes with DNA and to compete with native enzyme for the formation of rifampicin-resistant complex. The ratio of the complex formation constants for the native and modified enzyme was determined to be equal to 10. The enzyme modified to such extent loses the activity in DNA dependent RNA as well as pppApU synthesis. Vmax value rather than Km value for both ATP and UTP decreases following the modification reaction. Incubation of the enzyme modified to the 10% of residual activity with 0.2 M hydroxylamine for 2 hours results in restoration of RNA polymerase activity. Most but not all of the modified histidyl residues restore their native structure. Two of 13 histidyl residues were modified irreversibly due to Bamberger's cleavage reaction, but these two residues were found to be unessential for RNA polymerase activity. Reaction with higher concentration of the diethylpyrocarbonate induces modification of more than 15--20 histidyl residues and leads to irreversible inactivation of the enzyme. Nevertheless the modification of the additional histidyl redidues was reversible as well as the modification of the first 11 residues. RNA polymerase modified to such extent loses the ability to bind DNA. Preformation of the initiated ternary complex of RNA polymerase with template and product fails to protect the enzyme from reversible inactivation at a low reagent concentration, but markedly decreases the rate of the irreversible and unspecific modification of sulfhydryl or amino groups of the enzyme. Reaction with the ternary complex results in reversible inactivation of the enzyme with respect to elongation of RNA chains as well as the pyrophosphate exchange reaction. The complex itself was, however, completely stable under the reaction conditions and the enzyme subunit structure was also conserved after the reaction. Evidently, the mild modification of the histidyl residues with diethylpyrocarbonate selectively inhibits RNA chain elongation.
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PMID:[Modification of the RNA-polymerase of Escherichia coli by diethylpyrocarbonate]. 37 63

An enzyme has been isolated from Escherichia coli strains harboring the I-like plasmid R64drd11, which is capable of initiating DNA synthesis on the circular, single-stranded DNA of phages phi X174, fd, and G4. In the conversion of these templates to duplex forms in vitro, the enzyme can substitute for the functions of E. coli dna B-dnaB-dnaC-dnaG proteins, E. coli RNA polymerase, and E. coli dnaG protein, respectively. The enzyme requires all four ribonucleoside triphosphates for optimal activity, although a combination of ATP, CTP, and GTP can almost completely satisfy the rNTP requirement. The enzyme appears to cooperate specifically with DNA polymerases III because single-stranded DNA-dependent synthesis takes place in extracts deficient in DNA polymerases I and II but not in extracts from a dnaZ mutant. Highly purified enzyme preparations consist mostly of two major polypeptides, Mr 140,000 and 180,000, when analyzed by sodium dodecyl sulfate gel electrophoresis. These polypeptides cosediment with the enzyme activity through a glycerol gradient with a sedimentation coefficient of 3.6 S. DNA priming activity in extracts of E. coli strains harboring the mutant plasmids R64drd11 or ColIdrd1, which are derepressed in functions of conjugational DNA transfer, severalfold higher than the activity from strains carrying the corresponding wild-type plasmid. This correlation suggests that the enzyme may play a role in conjugational DNA synthesis.
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PMID:A DNA primase specified by I-like plasmids. 38 43

A new class of fluorescent nucleotide analogs which contain the fluorophore 1-aminonaphthalene-5-sulfonate attached via a gamma-phosphoamidate bond has been synthesized. Both the purine and pyrimidine analogs have fluorescence emission maxima at 460 nm. Cleavage of the alpha-beta-phosphoryl bond produces change in both the absorption and fluorescence emission spectra. The fluorescence of the pyrimidine analogs is quenched; cleavage of the alpha-beta-phosphoryl bond of the UTP analog produces about a 14-fold increase in fluorescence intensity at 500 nm. Under the same conditions the fluorescence of the CTP analog increases about 8-fold, whereas the fluorescence of the purine analogs shows only a slight change. These derivatives are good substrates for Escherichia coli RNA polymerase with only slightly increased Km values and with Vmax values about 50 to 70% that of the normal nucleotides. They are used less efficiently by wheat germ RNA polymerase II. The ATP analog can be used by E. coli RNA polymerase to initiate RNA chains.
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PMID:Synthesis and properties of fluorescent nucleotide substrates for DNA-dependent RNA polymerases. 38 81

The phi29 early mRNA's synthesized in infected Bacillus subtilis were studied by using sedimentation velocity analysis, polyacrylamide gel electrophoresis, and hybridization of phi29 DNA fragments generated by the restriction endonuclease Eco RI. Viral RNAs synthesized in vivo in the resence of chloramphenicol were found to hybridize to Eco RI-A, -C, and -D fragments, but not to Eco RI-B and -E fragments, of the viral genome. Major early mRNA sedimenting as 16S material in neutral sucrose gradients was examined in detail. Radioactive phi29 RNA, purified by sucrose gradient centrifugation, was hybridized to either the Eco RI-A or Eco RI-C DNA fragment. The RNA was eluted from the hybrids and then tested for complementary hybrid formation with Eco RI-A and -C fragments. RNA eluted from the Eco RI-A fragment annealed only to the Eco RI-A fragment and not to the Eco RI-C fragment. Similarly, RNA eluted from the Eco RI-C fragment hybridized to the Eco RI-C and -D fragments. Viral RNAs synthesized in vitro using B. subtilis RNA polymerase hybridized to both Eco RI-A and -C DNA fragments. Furthermore, RNA initiated with [gamma-(32)P]GTP also hybridized to both Eco RI-A and -C fragments. These results indicate that there are at least two efficient promotors for early transcription on the phi29 chromosome. In addition, a low-molecular-weight RNA initiated with [gamma-(32)P]ATP was found to hybridize exclusively with the Eco RI-A fragment. Kinetic studies of phi29 mRNA synthesis during the lytic cycle have shown that viral RNAs hybridizable to the Eco RI-A and -C fragments are synthesized immediately after phage infection. On the other hand, mRNA specific for the Eco RI-B fragment was not synthesized for several minutes after phage infection. Based on the results of the in vivo and in vitro transcription studies, a transcription map of the phi29 chromosome is proposed.
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PMID:Transcription of the genome of bacteriophage phi 29: isolation and mapping of the major early mRNA synthesized in vivo and in vitro. 40 15


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