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 inhibitor sensitivity and functional domains of recombinant encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase (3Dpol) have been extensively analyzed. The inhibitor profiles of EMC virus 3Dpol and Escherichia coli DNA-dependent RNA polymerase are distinct, and experiments with substrate analogs indicate that EMC virus 3Dpol lacks reverse transcriptase activity. Twenty amino acid substitutions were engineered in EMC virus 3Dpol based on sequence alignments of viral RNA-dependent RNA polymerases that identified conserved amino acid residues within motifs. Ten out of 17 conservative substitutions within the four most conserved motifs reduced the RNA polymerase activity of the mutants to 0-6% of the activity of the wild-type enzyme, demonstrating the importance of these amino acids in the structure and/or function of EMC virus 3Dpol. Remarkably, 5 of the 10 mutations in EMC virus 3Dpol which had the most drastic effect on its RNA polymerase activity (D240E, S293T, N302Q, G332A, and D333E) were found to correspond to active site residues in E. coli DNA-dependent DNA polymerase I (Klenow). Our results reveal that a basic structural and functional framework is conserved in the most distantly related classes of nucleic acid polymerases and demonstrate the validity of modeling the active site of an RNA-dependent RNA polymerase on the known structure of a DNA polymerase.
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PMID:Point mutations which drastically affect the polymerization activity of encephalomyocarditis virus RNA-dependent RNA polymerase correspond to the active site of Escherichia coli DNA polymerase I. 131 53

Carcinogen-induced expression of the integrated viral genome was examined on SV40-transformed Chinese hamster cells. Carcinogen treatment markedly increased the transcription rate and the steady state mRNA level of both early and late viral transcripts. Carcinogen-induced transcription was mediated by RNA polymerase II. The increase in viral gene expression was also detected at the protein level, although at a reduced amplitude. Enhanced transcription was apparent as early as 12 hr postexposure and was considerably elevated after 24-36 hr. The increased gene expression depended on the existence of a functional replication machinery, as indicated by two lines of evidence. First, a cell line that harbors origin-deleted SV40 failed to respond to carcinogen treatment by increasing transcription and expression of T antigen. Furthermore, carcinogen-induced overtranscription was inhibited by aphidicolin, an inhibitor of DNA polymerase alpha. The involvement of the replication apparatus in the enhanced expression points to mechanistic similarities between the carcinogen-induced viral gene expression in the drug-treated semipermissive cells and the SV40 lytic pathway under permissive conditions. It is therefore suggested that cellular permissivity to viral development is enhanced following exposure to carcinogens. The implications of these findings for the nature of cellular permissivity to viral infection and the synergistic effects of carcinogens and tumor viruses are discussed.
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PMID:Carcinogen-induced activation of SV40 gene expression in a semi-permissive environment. 132 84

Adducts produced by modification of DNA with benzo[a]pyrene diolepoxide (BPDE) are known to inhibit both DNA and RNA synthesis. This phenomenon has been used as a method for determining the distribution of carcinogen binding within defined DNA sequences. A critical comparison of different enzyme activities on adducted DNA is needed, since different enzymes may process adducted DNA differently. Thus, we compared blocks in DNA polymerase activity with that of an RNA polymerase and with an exonuclease at single base resolution. BPDE adducts blocked the progression of cloned T7 DNA polymerase (Sequenase) in a dose-dependent manner. Although the majority of these blocks were at one base prior to adducted guanines, we also observed some blocks opposite specific guanines, suggesting that in some sequences the polymerase inserted a base opposite the modified guanine. Digestion with T4 DNA polymerase (3'----5') exonuclease activity was also blocked in BPDE-adducted DNA; however, fragments produced by blocks in T4 exonuclease migrated two or more bases longer than the corresponding guanine. Mapping of adduct distributions using both Sequenase and T4 exonuclease gave similar results, demonstrating that a long tract of guanines was preferentially modified, and within a polyguanine sequence, the 5' guanines were more heavily modified than the 3' guanines. Transcription of adducted DNA by SP6 RNA polymerase was also inhibited in a dose-dependent manner. However, adducted bases which posed strong blocks to the DNA polymerase were not always strong blocks to the RNA polymerase. Thus, in terms of adduct distribution, Sequenase and T4 exonuclease provided more consistent results than the RNA polymerase, since blockage of the RNA polymerase correlated poorly with guanines.
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PMID:DNA polymerase, RNA polymerase and exonuclease activities on a DNA sequence modified by benzo[a]pyrene diolepoxide. 132 70

The genomic hypervariation of human immunodeficiency virus 1 (HIV-1) could result from misincorporations by the viral reverse transcriptase. We developed an assay for reverse transcriptase fidelity during RNA-dependent as well as DNA-dependent DNA polymerization in vitro. A lacZ alpha RNA fragment transcribed by T3 RNA polymerase was used to mimic first-strand reverse transcription. The corresponding DNA template was used to examine errors by reverse transcriptase during second-strand DNA synthesis. With both templates, the mutations introduced by reverse transcriptase were identified by their mutant phenotypes in an M13 lacZ alpha-complementation assay. We found that the reverse transcriptase from human immunodeficiency virus 1 (HIV-1 RT) was less accurate than the reverse transcriptase from Moloney murine leukemia virus (MLV RT) or the Klenow fragment of Escherichia coli DNA polymerase I (Pol I) on either RNA or DNA templates. The frequency of misincorporation by HIV-1 RT was 1 in 6900 nucleotides polymerized on the RNA template and 1 in 5900 on the DNA template. The error rates of MLV RT and Pol I on the RNA template were less than 1 in 28,000 and 37,000, respectively. The most frequent mutations produced by HIV-1 RT copying the RNA template were C----T transitions and G----T transversions resulting from misincorporation of dAMP.
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PMID:Fidelity of HIV-1 reverse transcriptase copying RNA in vitro. 137 Sep 10

A molecular cDNA clone (P1 KIN) was isolated that encodes the human RNA-dependent P1/eIF-2 alpha protein kinase. The complete cDNA sequence of the P1 KIN cDNA was determined; the longest open reading frame (ORF) encoded a 551 amino acid protein with a deduced molecular weight of 62055 Da. Transcripts prepared from the P1 KIN cDNA by transcription in vitro with T7 RNA polymerase programmed the cell-free synthesis of a protein indistinguishable by immunoprecipitation and immunoblot gel analyses from the authentic 67-kDa P1 protein synthesized in human U cells treated with interferon (IFN). Furthermore, by use of a sensitive primer extension assay with T7 DNA polymerase, the major site of translation initiation within the deduced ORF of the P1 KIN cDNA was directly identified. Northern RNA gel-blot analysis revealed that the P1 KIN cDNA strongly hybridized to two IFN-induced mRNAs present in both human amnion U cells and HeLa cells; their sizes were 2.5 and 6 kb. Both transcripts were efficiently induced by IFN-alpha, but poorly by IFN-gamma. Polyclonal antibody was prepared against the product of the P1 KIN cDNA expressed in Escherichia coli. In Western blot analysis the antibody recognized a 67-kDa protein induced in human cells by IFN-alpha and, in addition, a 90-kDa protein whose level was not greatly altered by IFN treatment. The IFN-induced 67-kDa protein was found associated with the ribosomal salt-wash fraction of IFN-treated human cells, whereas the 90-kDa protein was predominantly in the S100 soluble fraction. The time course for the induction by IFN-alpha of RNA-dependent protein P1 kinase activity measured by immunoprecipitation was comparable to the time course for protein P1 induction measured by Western immunoblot analysis. The amino acid sequence of P1/eIF-2 alpha protein kinase deduced from the cDNA was 62% identical with the 518-residue murine TIK kinase and contained, within the carboxy-terminal half of the protein, the motifs commonly conserved among protein-serine/threonine kinases. The amino-terminal half of the P1 protein did not possess conserved kinase motifs, but did show extensive homology with vaccinia virus-predicted protein E3L.
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PMID:Mechanism of interferon action: cDNA structure, expression, and regulation of the interferon-induced, RNA-dependent P1/eIF-2 alpha protein kinase from human cells. 137 53

In order to test the proposal that most nucleotide polymerases share a common active site structure and folding topology, we have generated 22 mutations of residues within motifs A, B and C of T7 RNA polymerase (RNAP). Characterization of these T7 RNAP mutants showed the following: (i) most of the mutations resulted in moderate to drastic reductions in T7 RNAP transcriptional activity supporting the idea that motifs A, B and C identify part of the polymerase active site; (ii) the degree of conservation of an amino acid within these motifs correlated with the degree to which mutation of that amino acid reduced transcriptional activity, supporting the predictive ability of this alignment in identifying the most functionally critical residues; (iii) a comparison of DNAP I and T7 RNAP mutants revealed similarities (as well as differences) between corresponding mutant phenotypes; (iv) the Klenow fragment structure is shown to provide a reasonable basis for interpretation of the differential effects of mutating different amino acids within motifs A, B and C in T7 RNAP. These observations support the proposal that these polymerase active sites have similar three-dimensional structures.
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PMID:Mutations in T7 RNA polymerase that support the proposal for a common polymerase active site structure. 139 70

The nucleotide sequence of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa, was determined. The termini of the 7-kb plasmid are 349-bp inverted repeats (TIRs). Each DNA strand contains a long open reading frame (ORF) which begins within the TIR and extends toward the centre of the plasmid. ORF-1 codes for a single-subunit RNA polymerase that is not closely related to that encoded by another Neurospora plasmid, kalilo. The ORF-2 product may be a B-type DNA polymerase resembling those encoded by terminal protein-linked linear genetic elements, including linear mitochondrial plasmids and linear bacteriophages. A separate coding sequence for the terminal protein could not be identified; however, the DNA polymerase of maranhar has an amino-terminal extension with features that are also present in the terminal proteins of linear bacteriophages. The N-terminal extensions of the DNA polymerases of other linear mitochondrial plasmids contain similar features, suggesting that the terminal proteins of linear plasmids may be comprised, at least in part, of these cryptic domains. The terminal protein-DNA bond of maranhar is resistant to mild alkaline hydrolysis, indicating that it might involve a tyrosine or a lysine residue. Although maranhar and the senescence-inducing kalilo plasmid of N. intermedia are structurally similar, and integrate into mitochondrial DNA by a mechanism thus far unique to these two plasmids, they are not closely related to each other and they do not have any nucleotide sequence features, or ORFs, that distinguish them clearly from mitochondrial plasmids which are not associated with senescence and most of which are apparently non-integrative.
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PMID:Genetic organization and structural features of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa. 142 26

We report on the discovery and isolation of DNA- and RNA-containing macromolecular nuclear complexes whose purified major DNA possessed electrophoretic mobilities of approximately 90 and approximately 25 kbp. The deoxyribonucleoprotein-ribonucleoprotein complexes contain RNA and DNA polymerase and primase activities and were isolated from nuclei of murine RAW117 large-cell lymphoma cells by restriction digestion with Msp-I, gentle extraction with solutions containing MgCl2, but without chelating agents, and low ionic strength gel electrophoresis. Two-dimensional (isoelectric focusing/M(r)) gel electrophoresis and silver staining of the proteins of the complexes after treatment with DNase I indicated the presence of approximately 30 protein components. In vitro DNA and RNA polymerase/primase assays showed that the DNP/RNP complexes had very high enzyme specific activities. Using the DNP/RNP complexes a discrete DNA polymerase alpha product of approximately 85 kbp was synthesized that was not synthesized in the presence of the DNA polymerase alpha inhibitor aphidicolin. RNA polymerase assays in the presence of excess alpha-amanitin indicated that the complexes possessed significant RNA polymerase I activity. Preparing the complexes at various times after the release of cells from a double thymidine block showed the complexes as well as the complex-associated enzyme activities to be cell-cycle dependent. The DNA and RNA polymerase-related activities were highest in late S phase, 7 and 9 h, respectively, after release from the double thymidine block. The complexes synthesized a specific in vitro DNA polymerase product using endogenous substrate and nucleotide precursors.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nucleoprotein complexes released from lymphoma nuclei that contain the abl oncogene and RNA and DNA polymerase and RNA primase activities. 142 73

Thirty-four mutants of phage T7 RNA polymerase (RNAP) were generated by linker-insertion mutagenesis and characterized with respect to their ability to carry out various steps in the transcription cycle. A number of mutants with interesting biochemical properties were identified. These include: (1) Mutant RNAPs that are catalytically active but that bind weakly to a T7 promoter; one of these mutants is affected in a region of the RNAP that exhibits homology with the sigma subunit of Escherichia coli RNAP. Another is affected in a region that has been previously implicated in the discrimination of T7 versus T3 promoters (Joho, et al., 1990). (2) Mutant RNAPs that can bind to the promoter but are transcriptionally inactive; some of these RNAPs lack catalytic activity, others are catalytically active but are unable to initiate productive transcription at a T7 promoter. Among the latter class of mutants are enzymes that appear to be weakened in their ability to melt open (or to remain associated with) double-stranded DNA; these RNAPs make only abortive initiation products and are unable to proceed to the formation of a productive elongation complex. The mutations causing this phenotype affect regions of the RNAP that exhibit homology with the catalytic site of DNA polymerase I (Delarue et al., 1990). (3) A C-terminal insertion mutant with properties similar to a previously characterized "foot" mutant (Mookhtiar et al., 1991). This RNAP appears to be defective in the very early steps of transcription and may be unable to translocate and/or empty the active site. (4) A mutant that is transcriptionally active, but is unable to complement the growth of T7 gene 1- phage. This phenotype may result from disruption of a function of the RNAP that is distinct from its role in RNA synthesis.
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PMID:Characterization of bacteriophage T7 RNA polymerase by linker insertion mutagenesis. 145 59

The DNA polymerase gene of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) was cloned and sequenced. The predicted DNA polymerase protein (1113 amino acids, 115.9K) was found to have an amino acid identity of 48% with the corresponding gene of the Autographa californica MNPV (AcMNPV). It contains five domains associated with substrate binding, primase interaction, and pyrophosphate hydrolysis and three domains associated with 3'-->5' exonuclease activity common to other DNA polymerases. A region with a conserved TATA promoter and a CAGT mRNA start site sequence motif was identified and shown to be transcribed by RNA polymerase II, indicating that the LdMNPV DNA polymerase gene is expressed as an early gene. An open reading frame possibly expressed as a late gene, oriented in the opposite direction and overlapping the N-terminal coding region of the DNA polymerase gene was found in the LdMNPV sequence and was shown to be conserved in the same position in AcMNPV.
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PMID:Characterization of the nucleotide sequence of the Lymantria dispar nuclear polyhedrosis virus DNA polymerase gene region. 146 55


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