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)

The imido ester, methyl acetimidate, which specifically amidinates lysine residues, modifies RNA polymerase core enzyme, leading to rapid loss of activity. Calf thymus DNA partially protects the enzyme against this inactivation, an effect which disappears at high salt concentration. DNA protects 17 +/- 6 lysines from amidination at low salt concentration. The dependence of amidination on methyl acetimidate concentration is examined in the presence of DNA at high and low salt concentration. Analysis of the data suggests a class of approximately 12 lysines which are protected by DNA, consistent with the above estimate. These lysines are approximately 5--10-fold more reactive than most other available lysine residues.
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PMID:Identification of a class of lysines within the non-specific DNA-binding site of RNA polymerase core enzyme from Escherichia coli. 680 29

One of the four subunits of bacteriophage Q beta RNA replicase is elongation factor Tu (EF-Tu), the host aminoacyl-tRNA (AA-tRNA) binding protein. To determine whether the RNA polymerase activity requires the tRNA binding site of EF-Tu, we reconstituted replicase with EF-Tu . GTP covalently bound to AA-tRNA. This cross-linked ternary complex (XLTC) was formed by the reaction of N epsilon-bromoacetyl-Lys-tRNA with EF-Tu-GTP. In an EF-Tu-dependent system for the reconstitution of replicase, XLTC restored polymerase activity at least as well as an equivalent amount of EF-Tu. Replicase reconstituted with XLTC was resolved from replicase containing EF-Tu by chromatography on phosphocellulose, a result which confirmed that the tRNA moiety was incorporated into the enzyme. Chromatographic analysis of reconstitution mixtures revealed that XLTC was incorporated into replicase as extensively as EF-Tu. From these results, it appears that the AA-tRNA binding site on EF-Tu is not required for the assembly or activity of Q beta RNA replicase. Furthermore, because the tRNA macromolecule is cross-linked to His-66 of the EF-Tu, the region surrounding His-66 must normally be exposed on the surface of the replicase.
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PMID:Transfer RNA cross-linked to the elongation factor Tu subunit of Q beta replicase does not inhibit Q beta RNA replication. 701 63

The catalytic center of wheat germ DNA-dependent RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) as a model eukaryotic enzyme system was probed with two purine nucleoside dialdehydes, 6-methylthioinosinedicarboxaldehyde (MMPR-OP) and a derivative 6-[(acetylaminoethyl)-1-naphthylamine-5-sulfonyl]thioinosinedicarboxaldehyde (AMPR-OP). Both drugs gave noncompetitive inhibition with respect to [3H]UMP incorporations into RNA, and inhibitor bindings were reversed with initiation substrates. The Ki values for MMPR-OP and AMPR-OP were determined to be 0.64 mM and 1.0 muM respectively. The drugs were covalently bound to the catalytic center by NaBH4 reduction. Both were found bound to the largest enzyme subunit, IIa. It is tentatively concluded that MMPR-OP and AMPR-OP inhibit RNA polymerase II by binding to an essential lysine in the initiation subsite of the catalytic center located on the IIa subunit.
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PMID:Probes of eukaryotic DNA-dependent RNA polymerase II-II. Covalent binding of two purine nucleoside dialdehydes to the initiation subsite. 713 56

Using immunofluorescent labeling and laser-scanning confocal microscopy, we show that isoforms of histone H4 acetylated on lysine 5, 8 and/or 12 (H4.Ac5-12), as well as RNA polymerase II, become enriched at the nuclear periphery around the time of zygotic gene activation, i.e., the 2-cell stage, in the preimplantation mouse embryo. In contrast, DNA and H4 acetylated on lysine 16 are uniformly distributed throughout the cytoplasm. Culture of embryos with inhibitors of histone deacetylase trichostatin A and trapoxin results in an increase in the (1) amount of acetylated histone H4 detected by immunoblotting, (2) intensity and sharpness of the peripheral staining for H4.Ac5-12, and (3) relative rate of synthesis of proteins that are markers for zygotic gene activation. The enhanced staining for H4.Ac5-12 at the nuclear periphery seems to require DNA replication, but appears independent of cytokinesis or transcription, since its development is inhibited by aphidicolin but not by either cytochalasin D or alpha-amanitin. Lastly, the restricted localization of H4.Ac 5-12 is not observed in the 4-cell embryo or at later stages of preimplantation development. These results suggest that changes in chromatin structure underlie, at least in part, zygotic gene activation in the mouse.
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PMID:Temporally restricted spatial localization of acetylated isoforms of histone H4 and RNA polymerase II in the 2-cell mouse embryo. 755 21

Direct chemical acetylation of an oligonucleosomal template for bacteriophage T7 RNA polymerase is accompanied by a substantial increase in its capability to support RNA synthesis. The template was assembled from a plasmid, containing a promoter and a terminator for T7 RNA polymerase, plus one (H3-H4)2 tetramer and two H2A.H2B dimers for each 200 base pairs of DNA. Under the employed conditions, acetylation modifies in a preferential way the lysine residues located in the amino-terminal domains of core histones. When the template is assembled with acetylated tetramers and untreated dimers, its efficiency in promoting RNA synthesis is also largely increased. Since a previous work reported transcriptional stimulation upon acetylation of H2A.H2B dimers [Puerta et al. (1995) Biochem. Biophys. Res. Commun. 210, 409], the transcriptional repression brought about by core histone octamers seems to require that the amino-terminal domains of both (H3.H4)2 tetramers and H2A.H2B dimers are not acetylated.
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PMID:Transcriptional properties of oligonucleosomal templates containing acetylated (H3-H4)2 tetramers. 763 40

Six recombinant Japanese encephalitis virus (JEV) isolates were recovered from infectious RNAs transcribed by T7 RNA polymerase from molecularly cloned cDNA templates. Three of the recombinant viruses had characteristics similar to the wild-type parent virus, JaOArS982. The other 3 recombinant viruses exhibited an attenuated phenotype in mice. An avirulent recombinant virus, IC47, was characterized and compared with the wild-type parent virus and a virulent recombinant virus, IC37. IC47 produced smaller plaques than parent or IC37 viruses and exhibited no viremia or neuroinvasion in young adult mice inoculated subcutaneously and no mortality when inoculated intracerebrally. IC47 was also immunogenic and protective in the murine model. The probable basis for attenuation, revealed by nucleotide sequence analysis, was a single amino acid substitution at position 138 (Glu to Lys) in the E protein.
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PMID:Characterization of a highly attenuated Japanese encephalitis virus generated from molecularly cloned cDNA. 775 89

Histone-DNA templates for bacteriophage T7 RNA polymerase were assembled from a plasmid containing a promoter and a terminator for T7 RNA polymerase, intact (H3.H4)2 tetramers, and either untreated or chemically acetylated H2A.H2B dimers. The nucleosomal particles containing acetylated H2A.H2B dimers protect 145 base pairs of DNA against micrococcal nuclease digestion and prevent the reaction with psoralen of 80 to 145 DNA base pairs. The inhibition of transcriptional initiation caused by the association of DNA with intact core histone octamers decreases significantly when the histone octamers contain acetylated H2A.H2B dimers. These results suggest a role for H2A.H2B dimers in the control of transcription, which might be mediated through acetylation and deacetylation of their lysine residues.
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PMID:Acetylation of histone H2A.H2B dimers facilitates transcription. 775 16

Poliovirus RNA polymerase (3Dpol) was cross-linked to [32P]ribonucleoside triphosphates (NTPs) by reduction of oxidized NTP-protein complexes. Cross-linked complexes were digested with cyanogen bromide, and resulting peptides were fractionated by reverse-phase HPLC. 32P-Labeled peptides were purified by secondary HPLC fractionation and/or additional digestion with endoproteinases Glu-C, TPCK-trypsin, or Asp-N followed by another HPLC fractionation. N-Terminal sequences of the major [32P]-peptides were determined, and approximate sizes of these peptides were obtained by SDS-polyacrylamide gel electrophoresis. Two major NTP binding sites in 3Dpol were found. One site was between Asp-266 and Met-286; possible binding residues in this fragment were Lys-276, Lys-278, or Lys-283. A second binding site was between Ala-57 and Met-74 with Lys-61 or Lys-66 as possible binding residues. Alignment of these regions on the known structure of HIV-1 reverse transcriptase allowed us to predict the position of the downstream nucleotide binding site in the conserved "fingers" subdomain present near the active site cleft of both RNA and DNA polymerases. The N-terminal nucleotide binding site is not contained within a region that is conserved among other polymerases.
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PMID:Identification of nucleotide binding sites in the poliovirus RNA polymerase. 775 55

The DNA-binding protein MetR belongs to the LysR family of transcriptional activators and is required for expression of the metE and metH promoters in Escherichia coli. However, it is not known if this activation is mediated by a direct interaction of MetR with RNA polymerase. In a search for RNA polymerase mutants defective in MetR-mediated activation of the metE gene, we isolated a mutation in the alpha subunit of RNA polymerase that decreases metE expression independently of the MetR protein. The mutation does not affect expression from the metH promoter, suggesting that the alpha subunit of RNA polymerase interacts differently at these two promoters. The mutation was mapped to codon 261 of the rpoA gene, resulting in a change from a glutamic acid residue to a lysine residue. Growth of the mutant is severely impaired in minimal medium even when supplemented with methionine and related amino acids, indicating a pleiotropic effect on gene expression. This rpoA mutation may identify either a site of contact with an as yet unidentified activator protein for metE expression or a site of involvement by the alpha subunit in sequence-specific recognition of the metE promoter.
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PMID:A mutation in the rpoA gene encoding the alpha subunit of RNA polymerase that affects metE-metR transcription in Escherichia coli. 783 82

The ada gene of Escherichia coli K-12 encodes the 39-kDa Ada protein, which consists of two domains joined by a hinge region that is sensitive to proteolytic cleavage in vitro. The amino-terminal domain has a DNA methyltransferase activity that repairs the S-diastereoisomer of methylphosphotriesters while the carboxyl-terminal domain has a DNA methyltransferase activity that repairs O6-methylguanine and O4-methylthymine lesions. Transfer of a methyl group to Cys-69 by repair of a methylphosphotriester lesion converts Ada into a transcriptional activator of the ada and alkA genes. Activation of ada, but not alkA, requires elements contained within the carboxyl-terminal domain of Ada. In addition, physiologically relevant concentrations of the unmethylated form of Ada specifically inhibit methylated Ada-promoted ada transcription both in vitro and in vivo and it has been suggested that this phenomenon plays a pivotal role in the down-regulation of the adaptive response. A set of site-directed mutations were generated within the hinge region, changing the lysine residue at position 178 to leucine, valine, glycine, tyrosine, arginine, cysteine, proline, and serine. All eight mutant proteins have deficiencies in their ability to activate ada transcription in the presence or absence of a methylating agent but are proficient in alkA activation. AdaK178P (lysine 178 changed to proline) is completely defective for the transcriptional activation function of ada while it is completely proficient for transcriptional activation of alkA. In addition, AdaK178P possesses both classes of DNA repair activities both in vitro and in vivo. Transcriptional activation of ada does not occur if both the amino- and carboxyl-terminal domains are produced separately within the same cell. The mutation at position 178 might interfere with activation of ada transcription by changing a critical contact with RNA polymerase, by causing a conformational change of Ada, or by interfering with the communication of conformational information between the amino- and the carboxyl-terminal domains. These results indicate that the hinge region of Ada is important for ada but not alkA transcription and further support the notion that the mechanism(s) by which Ada activates ada transcription differs from that by which it activates transcription at alkA.
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PMID:Alteration of lysine 178 in the hinge region of the Escherichia coli ada protein interferes with activation of ada, but not alkA, transcription. 786 1


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