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

---

Query: EC:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two newly synthesized pyrimidine derivatives were found to possess antiviral activity against Mengovirus in Fogh and Lund (FL) cells and in a cell-free system. The inhibitory effect on RNA-dependent RNA polymerase of Mengovirus-infected FL cells was assayed using 14C-UTP as precursor. Addition of 50 or 100 muM of the inhibitors in a cell-free system of crude enzyme and nucleoside triphosphate medium for 60 min incubation at 37 degrees C resulted in about 40 to 60% lower counting rates for drug-treated reaction mixtures. The analysis of the polymerase synthesis product (virus RNA extracted from the cell-free reaction mixture and deproteinized by the phenol-SDS method) was carried out by means of agarose-acrylamide gel electrophoresis. The main finding was a reduction of single-stranded Mengovirus RNA (RNase-sensitive and LiCl-precipitable). The rates of synthesis of the replicative intermediate (LiCl-precipitable) and the replicative form of RNA (LiCl-soluble) were not significantly influenced.
...
PMID:Effects of pyrimidine derivatives on RNA-dependent RNA polymerase of mengovirus-infected Fogh and Lund (FL) cells. 18 80

Adenosine (beta,gamma-imido)triphosphate (AMP-PNP) and guanosine (beta,gamma-imido)triphosphate (GMP-PNP) are analogs of ATP and GTP with non-hydrolyzable gamma-phosphates. Although both AMP-PNP and GMP-PNP were used in place of ATP and GTP by Escherichia coli RNA polymerase to transcribe vaccinia virus DNA, only GMP-PNP was used by the transcriptase present within vaccinia virus cores. AMP-PNP specifically prevented initiation of transcription, since RNA initiated in the presence of ATP, GTP, and CTP was subsequently elongated by incubating the washed cores in the presence of AMP-PNP, GTP, CTP, and UTP. The RNA formed in this manner, however, was (i) several times longer than normal transcripts, indicating a defect in chain termination and/or cleavage of nascent RNA, (ii) was not polyadenylylated (although free polyadenylic acid formed), and (iii) was not extruded from the virus cores. Nearest neighbor analysis demonstrated that AMP-PNP was incorporated adjacent to all four nucleotides, and hybridization to restriction endonuclease fragments of vaccinia virus DNA indicated that the high-molecular-weight RNA was transcribed from representative fractions of the entire genome. The possibility of a block in processing rather than or in addition to a block in chain termination was suggested by the cleavage of the high-molecular-weight RNA within the core after replacement of AMP-PNP with ATP. Cleavage of purified high-molecular-weight RNA by a soluble endoribonuclease extracted from vaccinia virus cores, however, was not dependent upon ATP, nor was it inhibited by AMP-PNP. The latter results suggest that AMP-PNP blocks a step preceding cleavage.
...
PMID:Multiple roles for ATP in the synthesis and processing of mRNA by vaccinia virus: specific inhibitory effects of adenosine (beta,gamma-imido) triphosphate. 69 Nov 15

Ribonucleic acid (RNA)-dependent RNA polymerase activity was demonstrated in the microsomal and ribosomal fraction from the spleen cells of immunized mice. The enzyme activity was solubilized by Triton X-100 from the fraction and partially purified by Biogel A 1.5 m column chromatography. The RNA-dependent RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RAN preparation (iotaRNA) as template made from the spleens of immunized mice but very low activity was found with an RNA preparation made from the spleens of normal mice. Incorporation of 3H-UTP markedly decreased in the presence of RNase but not in the presence of DNase. DNA preparations made from the spleens of immunized mice were inactive as template for this enzyme. The iotaRNA preparation was fractionated by sucrose density gradient centrifugation. A fraction corresponding to 12-13 S was most active as a template. It was followed by a fraction corresponding to 6-7 S. Sucrose gradient analysis of the 3H-UTP-labeled product was attempted. Some properties of this enzyme are described.
...
PMID:Ribonucleic acid-dependent ribonucleic acid polymerase in the immune response. 123 May 9

AIDS, caused by human immunodeficiency virus (HIV), is one of the world's most serious health problems, with current protocols being inadequate for either prevention or successful long-term treatment. In retroviruses such as HIV, the enzyme reverse transcriptase copies the single-stranded RNA genome into double-stranded DNA that is then integrated into the chromosomes of infected cells. Reverse transcriptase is the target of the most widely used treatments for AIDS, 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI), but resistant strains of HIV-1 arise in patients after a relatively short time. There are several nonnucleoside inhibitors of HIV-1 reverse transcriptase, but resistance to such agents also develops rapidly. We report here the structure at 7 A resolution of a ternary complex of the HIV-1 reverse transcriptase heterodimer, a monoclonal antibody Fab fragment, and a duplex DNA template-primer. The double-stranded DNA binds in a groove on the surface of the enzyme. The electron density near one end of the DNA matches well with the known structure of the HIV-1 reverse transcriptase RNase H domain. At the opposite end of the DNA, a mercurated derivative of UTP has been localized by difference Fourier methods, allowing tentative identification of the polymerase nucleoside triphosphate binding site. We also determined the structure of the reverse transcriptase/Fab complex in the absence of template-primer to compare the bound and free forms of the enzyme. The presence of DNA correlates with movement of protein electron density in the vicinity of the putative template-primer binding groove. These results have important implications for developing improved inhibitors of reverse transcriptase for the treatment of AIDS.
...
PMID:Structure of HIV-1 reverse transcriptase/DNA complex at 7 A resolution showing active site locations. 137 66

Poliovirus RNA encodes an RNA-dependent RNA polymerase, designated 3Dpol, that catalyzes the synthesis of both plus and minus strand viral RNA. This enzyme was purified to near homogeneity from poliovirus-infected HeLa cells, recombinant baculovirus-infected insect cells, and from Escherichia coli transformed with an expression plasmid containing poliovirus 3D sequences. The two recombinant expression systems produced significantly higher yields of active enzyme than could be attained from virus-infected HeLa cells. All preparations contained a 52-kDa protein, recognized by antisera raised against 3D expressed as a fusion protein in E. coli. Immunoreactive protein resolved into 3-4 species on isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional gels. Efforts to demonstrate that the multiple spots resulted from phosphorylation were negative. Furthermore, no evidence for autophosphorylation of purified 3Dpol was obtained. Purified 3Dpol from recombinant sources manifested the same specific activities as enzyme from poliovirus-infected HeLa cells in both a poly(A)-dependent poly(U) polymerase assay and a poliovirus RNA-dependent RNA polymerase assay. The products of the latter reaction reached the length of the template (7.5 kilobases) in 20-30 min, indicating an elongation rate of approximately 300 nucleotides/min at 30 degrees C. No products exceeded the length of the template. Intermediate length products were detected, which presumably resulted from pauses in transcription due to template structure. All transcription was dependent on primer. The kinetic parameters of all three purified enzyme preparations were the same; the Km for UTP was 2.4 +/- 0.1 microM in an RNA polymerase activity assay. Product formation was linear for up to 45 min, except for a 3-5-min lag before synthesis began. The lag was independent of enzyme concentration, and independent of the template used. The lag was eliminated by preincubating enzyme, template, primer, and three of the four nucleotide triphosphates, but not by preincubating any subset of these components. This suggested that a preinitiation complex must form as a prerequisite to RNA synthesis. Partially purified preparations of 3Dpol from the three sources showed significant differences in activities and products, including the appearance of primer-independent polymerase activity and production of dimer-length RNA products. These variable properties are likely due to different contaminating activities provided by the different cellular hosts, since upon further purification, all three enzymes exhibited identical properties.
...
PMID:Purification, characterization, and comparison of poliovirus RNA polymerase from native and recombinant sources. 166 Aug 94

The poliovirus RNA-dependent RNA polymerase required an oligouridylate primer or a HeLa cell protein (host factor) to initiate RNA synthesis on poliovirion RNA in vitro. The polymerase synthesized template-sized product RNA in the oligouridylate-primed reaction. In the host factor-dependent reaction, the largest product RNA synthesized by the polymerase was twice the size of the template RNA. About half of the product RNA recovered from this reaction was shown to exist in the form of a snapback sequence. Time-course reactions and pulse-chase experiments showed that the product RNA was only slightly larger than the template RNA at early reaction times and that with time it increased in size to form the dimer-sized product RNA. Inhibition of the elongation reaction by adding only [alpha-32P]UTP and ATP resulted in the formation of template-sized product RNA. The dimer-sized product RNA was unaffected by phenol extraction or proteinase K treatment but was converted to template-sized molecules by S1 nuclease. Dimer-sized poliovirus RNA that was sensitive to S1 nuclease was also isolated from poliovirus-infected cells. The results from this study indicate that the labeled negative-strand product RNA synthesized in vitro was covalently linked to the positive-strand template RNA. Thus, in vitro, the primer-dependent poliovirus RNA polymerase may initiate RNA synthesis in the presence of the host factor by using the 3' end of the template RNA as a primer.
...
PMID:Poliovirus RNA-dependent RNA polymerase and host cell protein synthesize product RNA twice the size of poliovirion RNA in vitro. 298 94

Indomethacin blocks the biosynthesis of vesicular stomatitis virus (VSV) at the level of primary transcription, RNA replication, and protein synthesis (P. K. Mukherjee and R. W. Simpson (1985), Virology 140, 188-191). Nucleocapsids of infecting virus particles recovered from indomethacin-treated cells were analyzed for in vitro transcriptase activity. Incorporation of [3H]UTP in mixtures containing nucleocapsids from HEp-2 cells pretreated with 10(-3) M indomethacin was inhibited approximately 80% compared to control reactions containing nucleocapsids from untreated infected cells. The level of inhibition of in vitro transcriptase activity of viral nucleocapsids from drug-treated cultures varied according to the cell line used for infection. After indomethacin removal, cells regained their ability to produce enzymatically competent viral-transcribing complexes unless they were subsequently exposed to metabolic inhibitors such as actinomycin D or alpha-amanitin. Enzymatically defective nucleocapsids from indomethacin-treated cells showed enhanced in vitro transcriptase activity in the presence of modulators of prostaglandins and cyclic nucleotides. Electrophoretic analysis of product from in vitro transcriptase reactions revealed that these defective nucleocapsids are unable to synthesize VSV messenger RNA or normal size leader RNA species but only smaller transcripts of undetermined identity.
...
PMID:Transcriptionally defective nucleocapsids of vesicular stomatitis virus from cells treated with indomethacin. 302 67

RNA-dependent RNA polymerase (RDRP) activity was characterized in a cytoplasmic extract of Kunjin virus-infected Vero cells at 24 hr. The activity was influenced, possibly indirectly, by the length of prior treatment of infected cells with actinomycin D; however, 6 micrograms/ml actinomycin D and 10(-5) M alpha-amanitin in the RDRP assay had no effect. The replication complex was membrane-bound and Mg2+ was essential for RDRP activity. Incorporation was more dependent on exogenous UTP and GTP than ATP or CTP. The specific activity was low, and rate of incorporation of GMP decreased as the period of assay was increased; however, incorporation of label lasted for at least 60 min. RNA products were fractionated by LiCl precipitation, and kinetic studies showed that the sequence of accumulation of label was the same as that observed in vivo, viz., RI----RF----44 S RNA; limited reinitiation was also observed. This sequence of labeling also indicated that the in vitro RDRP activity was due to an enzyme capable of elongation, release, and reinitiation of Kunjin RNA synthesis and not merely end labeling or elongating preexisting RNA molecules. No labeled bands in urea-polyacrylamide gels were observed using extracts from mock-infected cells and hence the three RNA products of assays were readily identified in a single gel. The replication complex was still active after treatment with nonionic detergent, but no labeled 44 S RNA was detected in gels, even in the presence of RNasin in the assay which inhibited some nuclease activity. Antibodies to flavivirus-specific nonstructural proteins were preincubated with infected cell extracts in the presence and absence of detergent but no inhibition of RDRP activity was observed. However, anti-dsRNA plus detergent blocked activity by as much as 78% and label was found only in RF.
...
PMID:Characterization of Kunjin virus RNA-dependent RNA polymerase: reinitiation of synthesis in vitro. 302 75

A cytoplasmic, microsomal bound RNA-dependent RNA polymerase has been purified 2500-fold from rabbit reticulocyte lysates. The synthesis of RNA with the purified enzyme is absolutely dependent on the addition of an RNA template. The best template is hemoglobin messenger RNA, while bacteriophage RNA and poly(A,G) are less active, and DNA is completely inactive as a template. With poly(A,G) as a template, only UTP and CTP are incorporated into polynucleotide chains, indicating that the RNA polymerase is an RNA replicase and not a terminal transferase. With messenger RNA as a template, all four ribonucleoside triphosphates are required for maximal activity. The RNA-dependent RNA polymerase reaction is extremely sensitive to low concentrations of heme, rifamycin AF/013, and ribonuclease and resistant to actinomycin D and DNase. The discovery of RNA-directed RNA synthesis in reticulocytes offers an additional site for control of gene expression in mammalian cells and provides a possible mechanism for amplification of the expression of specific genes.
...
PMID:Reticulocyte RNA-dependent RNA polymerase. 451 33

To identify the initial steps of vesicular stomatitis virus transcription, we reconstituted purified nucleocapsid template with solubilized transcriptase and characterized the in vitro products of de novo transcription. In the absence of UTP and GTP, only leader gene products were synthesized; mRNA oligonucleotides were detected only after transcription of full-length leader was permitted. These data suggest that vesicular stomatitis virus polymerase does not enter the genome independently at each gene, but each polymerase begins transcription at the 3' end of the genome, and reaches internal genes only by sequentially transcribing the 3' preceding sequences. These results are consistent with the conclusion that the observed sequential transcription of vesicular stomatitis virus mRNAs is due to obligatory entrance of all polymerases at the leader gene, and suggest that the transcriptase and replicase may recognize the same promoter.
...
PMID:Reconstitution studies detect a single polymerase entry site on the vesicular stomatitis virus genome. 629 77


1 2 3 4 5 6 7 Next >>

---