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 effects of 4'-epi-daunorubicin, 4'-epi-adriamycin, and the corresponding beta anomers on the in vitro activity of Escherichia coli DNA polymerase I and RNA polymerase were determined and compared with the effects of the parent compounds. The observed effects parallel the cytotoxic activities, assayed by inhibition of mouse embryo fibroblast proliferation, and the inhibitory activities on DNA synthesis in cultured cells. The data indicate that the inverted configuration at position 1 of the amino sugar results in a markedly reduced biological activity. This conclusion is also substantiated by the data obtained with the beta anomer of adriamycin. A preliminary investigation on the binding properties of these derivatives suggests that the inverted configuration at C-1' produces a significant decrease in the binding to DNA. In contrast, epimerization at position 4' did not produce any significant change in activity. The relationship between biological and biochemical activity and DNA binding properties of the tested compounds are discussed with particularly reference to antitumor activity.
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PMID:Relationship between activity and amino sugar stereochemistry of daunorubicin and adriamycin derivatives. 77 33

A series of plasmids containing hepatitis A virus (HAV) cDNA was constructed such that positive-strand HAV RNA could be transcribed with T7 RNA polymerase. The plasmids differed in the number of 5'-terminal nucleotides representing the junctions between vectors and HAV sequences that were present in the transcripts. When these transcripts were used to transfect cultured BS-C-1 cells, it was found that only those transcripts that contained all of the 5'-terminal HAV nucleotides, in addition to one or more nucleotides from the vector, were capable of initiating an infectious cycle leading to production of progeny virus. Transcripts that contained one 5'-terminal nucleotide from the vector sequence but were missing two uridylate residues corresponding to the first two nucleotides of HAV sequences, or were missing U and C residues corresponding to nucleotides 2 and 3 of the HAV sequence, were not infectious. A similar plasmid containing poliovirus cDNA was engineered to produce transcripts similarly lacking the first two uridylate residues of the poliovirus RNA sequence. These transcripts were infectious.
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PMID:The 5'-terminal nucleotides of hepatitis A virus RNA, but not poliovirus RNA, are required for infectivity. 185 50

Ten of 12 Chandipura virus tdCE mutants, which exhibit temperature-dependent restriction of growth in chick embryo (CE) cells but not in BS-C-1 cells, showed deficient transcriptase activity in vitro at 39 degrees C relative to wild-type virus. A gradation in transcriptional activity at 39 degrees C in vitro was observed. Reversion of the tdCE phenotype to unrestricted growth in CE cells at 39 degrees C was accompanied by partial restoration of normal transcriptase activity at 39 degrees C, suggesting that reversion was mediated by either extragenic or intragenic suppression. Viral protein synthesis was reduced or absent in CE cells at 39 degrees C indicating that transcription was also defective in vivo under these conditions. Induction of heat-shock proteins in CE cells at 39 degrees C occurred normally in tdCE mutant-infected cells and RNA methylation in vitro was unaffected.
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PMID:In vitro transcriptase deficiency of temperature-dependent host range mutants of Chandipura virus. 242 18

The rrnB P1 promoter of Escherichia coli (starting sequence C-4-A-3-C-2-C-1-A+1-C+2-U+3-G+4) forms a binary complex with RNA polymerase that is highly unstable and requires the presence of transcription substrates ATP and CTP for stabilizing the enzyme-DNA association (Gourse, R. L. (1988) Nucleic Acids Res. 16, 9789-9809). We show that in the absence of UTP and GTP the stabilization is accomplished by short RNA oligomers synthesized in an unusual "-3-->" mode whereby the primer initiated at the +1 site presumably slips back by three nucleotides into the -3 site and is then extended yielding stable ternary complexes. By contrast, short oligomers initiated in the conventional "+1-->" mode without slippage do not exert the stabilization effect and are readily aborted from the promoter complex. The stable -3-->ternary complexes carry sigma factor but otherwise resemble elongation complexes in their high salt stability and in the fact that they are formed with a mutant RNA polymerase deficient in promoter binding. A model is proposed explaining the stability of the -3-->ternary complexes by RNA slipping into a putative "tight RNA binding site" in RNA polymerase which is normally occupied by RNA during elongation.
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PMID:Two modes of transcription initiation in vitro at the rrnB P1 promoter of Escherichia coli. 822 74

Hepatitis A virus (HAV) cDNAs encoding the P3 region proteins were expressed in vivo and in vitro to characterize the HAV 3D protein and to identify the cleavage site between 3C and 3D. Protein coding sequences were placed under control of a T7 promoter and an EMCV translational initiation signal. T7 RNA polymerase was provided by simultaneous infection of transfected BS-C-1 cells with a recombinant vaccinia virus vTF7-3 (T. R. Fuerst et al., Proc. Natl. Acad. Sci. USA 83, 8122-8126, 1986). Efficient synthesis and processing of P3 proteins occurred to yield 3CD (78 kDa), 3D (54 kDa), 3ABC (33 kDa), 3BC (25 kDa), and 3C (23 kDa). Similar products were produced by translation of T7 transcripts in a rabbit reticulocyte lysate in vitro. The 3C/D cleavage site was mapped by comparing the mobility of 3D in SDS-PAGE with 3D proteins engineered to begin at each of the two proposed cleavage sites; in addition, direct N-terminal sequencing of radiolabeled 3D protein from translation in vitro was performed. The results showed that 3D was formed by cleavage at the glutamine-arginine (Q/R) pair at position 1738 and 1739 of the HAV polyprotein. HAV 3D protein produced by autocatalytic cleavage of P3 precursor proteins in BS-C-1 cells is virtually completely insoluble and sediments after low-speed centrifugation. This is in contrast to the poliovirus 3D protein, produced from a similar construct, a significant portion of which remains soluble. Extracts containing the poliovirus 3D protein manifested high levels of RNA-dependent RNA polymerase activity, whereas those containing the HAV 3D protein showed no detectable activity by the same assay.
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PMID:Expression of hepatitis A virus precursor protein P3 in vivo and in vitro: polyprotein processing of the 3CD cleavage site. 829 Dec 34

Mutations in the 5' nontranslated RNA (5'NTR) of an attenuated, cell culture-adapted hepatitis A virus (HAV), HM175/P16, enhance growth in cultured African green monkey kidney (BS-C-1) cells but not in fetal rhesus monkey kidney (FRhK-4) cells (S. P. Day, P. Murphy, E. A. Brown, and S. M. Lemon, J. Virol. 66: 6533-6540, 1992). To determine whether these mutations enhance cap-independent translation directed by the HAV internal ribosomal entry site (IRES), we compared the translational activities of the 5'NTRs of wild-type and HM175/P16 viruses in two stably transformed cell lines (BT7-H and FRhK-T7) which constitutively express cytoplasmic bacteriophage T7 RNA polymerase and which are derived from BS-C-1 and FRhK-4 cells, respectively. Translational activity was assessed by monitoring expression of a reporter protein, chloramphenicol acetyltransferase (CAT), following transfection with plasmid DNAs containing bicistronic T7 transcriptional units of the form luciferase-5'NTR-CAT. In both cell types, transcripts containing the 5'NTR of HM175/P16 expressed CAT at levels that were 50- to 100-fold lower than transcripts containing the IRES elements of Sabin type 1 poliovirus or encephalomyocarditis virus, confirming the low activity of the HAV IRES. However, in BT7-H cells, transcripts containing the 5'NTR of wild-type virus. This translational enhancement was due to additive effects of a UU deletion at nucleotides 203 and 204 and a U-to-G substitution at nucleotide 687 of HM175/P16. These mutations did not enhance translation in FRhK-T7 or Huh-T7 cells (a T7 polymerase-expressing cell line derived from human hepatoblastoma cells) or in vitro in rabbit reticulocyte lysates. These results demonstrate that mutations in the 5'NTR of a cell culture-adapted HAV enhance viral replication by facilitating cap-independent translation in a cell-type-specific fashion and support the concept that picornaviral host range is determined in part by differences in cellular translation initiation factors.
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PMID:Mutations within the 5' nontranslated RNA of cell culture-adapted hepatitis A virus which enhance cap-independent translation in cultured African green monkey kidney cells. 855 62

The vaccinia virus/bacteriophage T7 expression system was adapted to Chinese hamster ovary (CHO) cells. Vaccinia virus undergoes abortive infection in CHO cells, which is characterized by a sharp reduction in protein synthesis at the stage of viral intermediate gene expression. We determined that expression of a T7 promoter-regulated chloramphenicol acetyltransferase gene was at least 20 times more efficient in permissive BS-C-1 than in CHO cells. The encephalomyocarditis virus 5'-untranslated region, which confers cap-independent translatability to mRNA, stimulated recombinant protein synthesis by 10-fold in both cell lines, maintaining the advantage of the BS-C-1 cells over CHO cells. Since the cowpox virus hr gene overcomes vaccinia virus host range restriction in CHO cells, we constructed a recombinant virus that carries an intact hr gene in addition to the T7 RNA polymerase gene. With this virus, synthesis of T7 RNA polymerase was enhanced and production of a recombinant protein occurred in CHO cells at the level observed in permissive cell lines. Extension of the vaccinia virus/bacteriophage T7 expression system to CHO cells should be of wide interest, as these cells have advantages for preparation of recombinant proteins in research and biotechnology.
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PMID:Recombinant protein synthesis in Chinese hamster ovary cells using a vaccinia virus/bacteriophage T7 hybrid expression system. 866 85

We describe a plastid in vitro transcription system that reflects characteristic features of the light-regulated transcription observed in vivo. Multiple transcripts of the wheat (Triticum aestivum) psbD/C gene cluster comprise six distinct 5[prime] ends including four transcription initiation sites designated as D/C-1 through D/C-4. Transcripts from one particular site, D/C-3, were found to be conspicuously enhanced in abundance after 4 h of illumination in vivo. The plastid extract prepared from 5-d-old dark-grown wheat seedlings was capable of transcribing from the D/C-2 and D/C-4 sites in vitro but had almost no transcription activity from the light-responsive D/C-3 site (the D/C-1 site was not examined). The plastid extract from 4-h-illuminated seedlings initiated transcription from the light-responsive site (D/C-3). Transcription from the D/C-2 and D/C-4 sites was not enhanced by using the extract from 4-h-illuminated seedlings, indicative of specific activation of the light-responsive promoter on the D/C-3 site by the extract from 4-h-illuminated seedlings. The plastid extract from 4-h-illuminated seedlings was divided into two fractions on a heparin-Sepharose column, into which the light-induced component(s) responsible for activation of the D/C-3 promoter and RNA polymerase were separated. The fraction containing the component(s) activating the D/C-3 promoter induced the transcription activity from the D/C-3 site in the plastid extract from dark-grown seedlings. It is concluded that the plastid extract from 4-h-illuminated seedlings contains some light-regulatory component(s) that activate specifically the light-responsive promoter.
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PMID:In Vitro Analysis of Light-Induced Transcription in the Wheat psbD/C Gene Cluster Using Plastid Extracts from Dark-Grown and Short-Term-Illuminated Seedlings. 1223 65

As part of a series of studies to discover new HIV reverse-transcriptase inhibitors, various novel 6alpha- and 6beta-naphthylthio 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio) thymine (HEPT) derivatives were synthesized, and in vitro anti-HIV-1 activity was evaluated. The results revealed that most of 6alpha-naphthylthio HEPT derivatives (7a-w) showed good activity [for 7e, IC50 value of 0.048 microM and selectivity index (SI) value of 735; for 7h, IC50 value of 0.057 microM and SI value of 579; for 7k, IC50 value of 0.063 microM and SI value of 565], 6beta-naphthylthio HEPT derivatives (8a-f) showed low activity, but the introduction of alpha nitro group to the C-1 position of the 6beta-naphthyl ring in the 6beta-naphthylthio series (11a-c) resulted in a dramatic increase in anti-HIV-1 activity.
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PMID:Nonnucleoside HIV-1 reverse-transcriptase inhibitors, part 5. Synthesis and anti-HIV-1 activity of novel 6-naphthylthio HEPT analogues. 1607 14

A novel class of HCV NS5B RNA dependent RNA polymerase inhibitors containing 2,3,4,9-tetrahydro-1H-carbazole and 1,2,3,4-tetrahydro-cyclopenta[b]indole scaffolds were designed and synthesized. Optimization of the aromatic region showed preference for 5,8-disubstitution pattern in both the scaffolds examined while favoring the n-propyl moiety for the C-1 position. 1,2,3,4-tetrahydro-cyclopenta[b]indole scaffold was slightly more potent than the corresponding 2,3,4,9-tetrahydro-1H-carbazole and analogue 36 displayed an IC50 of 550 nM against HCV NS5B enzyme.
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PMID:Design and synthesis of 2,3,4,9-tetrahydro-1H-carbazole and 1,2,3,4-tetrahydro-cyclopenta[b]indole derivatives as non-nucleoside inhibitors of hepatitis C virus NS5B RNA-dependent RNA polymerase. 1648 Aug 69


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