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)

Partial purification of the encephalomyocarditis protease synthesized in extracts from rabbit reticulocytes shows that the activity responsible for cleaving coat precursor protein cosediments with a previously unmapped virus-coded protein with an apparent molecular weight of 20,000. Tryptic analysis shows that this protein is derived from protein D, a virus-coded component of the encephalomyocarditis RNA polymerase.
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PMID:Protease required for processing picornaviral coat protein resides in the viral replicase gene. 22 66

When Semliki Forest virus ts-4 mutant infected cultures are grown at the permissive temperature (28 degrees C) and shifted to the restrictive temperature (39 degrees C), two different defects in RNA synthesis are manifested: (i) the synthesis of 26S RNA is stopped within 60 min (Saraste et al. 1977) and (ii) the increase in RNA synthesizing activity ceases, in contrast to cultures maintained at 28 degrees C, indicating that no new active RNA polymerase is formed at 39 degrees C. Accumulation of a non-structural precursor protein with an apparent mol. wt. of about 220 000 (ns220) was demonstrated in ts-4 infected cultures shifted to 39 degrees C. NS220 was labelled during short pulses given immediately after release of protein synthesis from hypertonic initiation block, suggesting that genes coding for ns220 are located near the initiation site at the 5'-end of the 42S RNA. The viral specificity of ns220 was shown by its disappearance after a shift to 28 degrees C and by labelling in the presence of sucrose, when no host cell protein synthesis is detectable. The two functional defects can be explained if the polypeptides responsible for the RNA polymerizing activity and that responsible for the synthesis of 26S RNA are components of the same non-structural polyprotein. A mutation in the latter polypeptide which prevents cleavage of the polyprotein would thereby prevent the further formation of active RNA polymerase. If cleavage of the polyprotein has taken place at the permissive temperature, the RNA polymerase would remain active also at 39 degrees C, whereas the polypeptide responsible for 26S RNA synthesis would become inactive due to the mutation.
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PMID:Cleavage defect in the non-structural polyprotein of Semliki Forest virus has two separate effects on virus RNA synthesis. 66 Jan 63

Previously we have shown that nuclear extracts from mouse cells contain a heterogeneous group of polypeptides (p65, p80, p90, p100) which form distinct DNA-protein complexes on the 18 base-pair sequence element (termed Sal-box), which constitutes the murine rDNA transcription termination signal. These distinct proteins mediate cessation of RNA polymerase I (pol I) transcription elongation and release of the nascent RNA chains, indicating that they function as termination factor(s). Here, we report the biochemical analysis of the pol I-specific transcription termination factor TTFI. We show that the heterogeneity of TTFI is due to limited proteolysis of a larger, 130 kDa precursor protein (p130). The DNA-binding activity of p130 is strongly reduced as compared to the proteolytic derivatives, indicating that the DNA-binding domain is repressed within the full-length molecule. We have used limited proteolysis to purify and functionally characterize a TTFI core polypeptide (p50) which still specifically binds to the Sal-box target sequence and directs rDNA transcription termination. The equilibrium constant of purified p50 to bind specifically to DNA is 9 x 10(9) M-1. Additionally, we demonstrate that TTFI binds to DNA as a monomer and that binding induces DNA bending. This observation suggests that not only specific DNA-protein and protein-protein interactions but also conformational alterations of DNA may play a role in the termination process.
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PMID:Limited proteolysis unmasks specific DNA-binding of the murine RNA polymerase I-specific transcription termination factor TTFI. 140 80

The Semliki-Forest-virus-specific nonstructural proteins are translated as a large polyprotein (2431 amino acid residues), from which the mature polymerase components nsP1, nsP2, and nsP4 are released by proteolytic cleavages. The complete ns polyprotein (P1234) can be cleaved in two alternative ways yielding either P123 (with sequences of nsP1, nsP2 and nsP3) and nsP4 or P12 (nsP1 plus nsP2) and P34 (nsP3 plus nsP4). We studied the possible autoproteolytic role of nsP4 involved in the cleavage between nsP3 and nsP4 in an in vitro transcription-translation system. cDNAs encoding P34 precursor and shorter precursor protein segments covering the nsP3-nsP4 cleavage region, were cloned under the T7 RNA polymerase promoter. The mRNAs synthesized in vitro were capped and translated in rabbit reticulocyte lysates. The translational products were analyzed by SDS/PAGE. The precursor proteins containing nsP4 sequences were cleaved yielding the products with expected sizes, indicating that the cleavage took place at the nsP3-nsP4 junction. By deleting and truncating the cDNA coding for nsP4, the proteolytic activity was mapped within the 102 amino-terminal amino acids of nsP4. The cleavage between nsP3 and nsP4 can be inhibited by pepstatin A and probably takes place in cis, since exogenously added nsP4 was unable to mediate it.
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PMID:The Semliki-Forest-virus-specific nonstructural protein nsP4 is an autoproteinase. 213 9

The ability of papaverine to inhibit human immunodeficiency virus (HIV) replication in H9 cell line and in peripheral blood mononuclear cell (PBMC) culture was examined. HIV-infected H9 cells were exposed to different concentrations of papaverine for 20 days. Reverse transcriptase (RT) activity and the presence of p24 in the supernatant were determined to assess the level of viral replication in treated and control cultures. The most effective concentration of papaverine in the culture medium was 10 micrograms/ml, a dose that did not significantly affect cell proliferation. At this drug concentration the treatment resulted in no RT activity or p24 expression in the supernatant and no virus antigen detection at the cellular level as demonstrated by Western blot (WB) analysis. The activity of the drug occurred in a short period of time (60 hours) as shown by radioimmunoprecipitation (RIP) assay and affected the synthesis of the env precursor protein gp160. The drug was also effective in inhibiting HIV replication in PBMC cultures and influenced specific viral markers, namely, RT and p24. Evidence of the efficacy of papaverine treatment was enforced by the finding in the treated PBMC cultures, compared with the untreated ones, of a reduced percentage of cells forming syncitia and of the inhibition of the virus-induced decrease in the number of cells. When an equal number of virus-infected H9 cells exposed or unexposed to papaverine was analyzed for HIV-specific proteins, a marked decrease in the expression of the viral proteins was observed in the treated cultures. At the same time, one cellular protein of molecular weight 69,000 was not inhibited by papaverine. This may indicate that, at least for one protein, synthesis may not be affected by the drug. Our data suggest that papaverine merits attention as a possible nontoxic candidate for the treatment of HIV infection.
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PMID:Inhibitory effect of papaverine on HIV replication in vitro. 271 67

Antibody to the poliovirus genome-linked protein, VPg, specifically immunoprecipitated the product RNA synthesized in vitro by the poliovirus RNA polymerase and HeLa cell host factor when VPg-linked poliovirion RNA was used as a template. The largest product RNA that was immunoprecipitated was twice the size of the template RNA. The complete denaturation of the product RNA with CH3HgOH had no effect on the immunoprecipitation reaction. In contrast, CH3HgOH denaturation prevented the immunoprecipitation of the oligo(U)-primed product RNA. Immunoprecipitation of the product RNA synthesized in the host-factor-dependent reaction was prevented if VPg was removed from the template RNA by pretreatment with proteinase K or if an RNA template without VPg was used in the reaction. The results support our previous evidence that a covalent linkage exists between the labeled negative-strand product RNA and the VPg-linked template RNA and suggest that the purified polymerase and host factor initiated RNA synthesis in vitro in the absence of VPg or a VPg-precursor protein.
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PMID:Anti-VPg antibody precipitation of product RNA synthesized in vitro by the poliovirus polymerase and host factor is mediated by VPg on the poliovirion RNA template. 300 84

Repetitious gene cassettes that encode the consensus decapeptide repeat of Mytilus edulis bioadhesive protein were designed, constructed, and expressed in Escherichia coli. The bioadhesive precursor (BP) with a relative molecular mass of 25,000 was expressed from one 600-bp gene at levels approaching 60% of total cell protein in strains employing T7 RNA polymerase for induction and carrying a repetitious gene comprised of a 30-bp unit repeat that accounts for E. coli codon bias. BP forms intracellular inclusions and yet methionine was processed from the N-terminus of the purified protein, as shown by amino acid composition and N-terminal sequencing, to give an authentic consensus precursor protein.
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PMID:Cloning, expression, and characterization of a synthetic analog to the bioadhesive precursor protein of the sea mussel Mytilus edulis. 776 30

Processing and assembly of bovine leukaemia virus-like particles were studied in African green monkey kidney cells using recombinant vaccinia viruses (rVVs) expressing regions of the bovine leukaemia virus genome. Unprocessed gag precursor protein (Pr44) was detected in immunoblot analysis of lysed cells and particles sedimented from culture supernatants after infection with a rVV carrying the gag and truncated protease (pro) gene. Processing of Pr44 was observed after infection of cells with a rVV carrying the gag and pro gene or a rVV expressing the gag, pro and polymerase (pol) gene. Reverse transcriptase activity was detected only in association with particles produced by gag-, pro- and pol-expressing recombinants. Thin section electron microscopic analysis of infected cells and pelleted particles revealed that Pr44 and processed gag proteins assembled at the cell membrane. Pr44 was released into the cell culture media as immature virus-like particles, whereas processed gag proteins from rVVs expressing gag and pro or gag, pro and pol formed mature particles.
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PMID:Retrovirus-like particles produced by vaccinia viruses expressing gag-pro-pol region genes of bovine leukaemia virus. 807 21

We have constructed a clone encoding the foot-and-mouth disease virus (FMDV) 3C protease gene (p3C) using the polymerase chain reaction. The construct was engineered to contain initiation and termination codons and cloned into a plasmid under the control of the bacteriophage T7 promoter. The p3C gene was expressed both in an in vitro transcription-translation system and in vivo in an Escherichia coli system containing an inducible T7 RNA polymerase gene. In both systems the expressed products were of the appropriate molecular weight and immunologically reactive with bovine convalescent serum. E. coli-expressed 3C protein was mainly found in the insoluble fraction of cell lysates. The E. coli-expressed protease was assayed in an in vitro system with radiolabeled P1 capsid precursor protein and P2 precursor protein as substrates. E. coli-expressed 3C completely processed the P1 and P2 precursors into mature capsid and nonstructural proteins, respectively. The kinetics of processing of P1 by E. coli-expressed 3C revealed the following order of cleavage: VP3-VP1, VP0-VP3, VP1-2A.
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PMID:Characterization of the foot-and-mouth disease virus 3C protease expressed in Escherichia coli. 821 67

Most organisms respond to heat by substantial alteration of the pattern of gene expression. This has been particularly well studied with Escherichia coli although the response has by no means been completely characterized. Here we report the characterization of 26 new heat shock genes of E. coli, termed hsl, discovered by global transcription analysis with an overlapping lambda clone bank. We have measured the molecular weights of the corresponding heat shock proteins and mapped each of them to within a few kilobases on the E. coli genome. In vitro, 16 of them can be activated by the E sigma 32 RNA polymerase, which specifically transcribes heat shock genes. In vivo expression kinetics of seven of eight examined new proteins were found to be similar to those of the four most studied heat shock proteins, DnaK, DnaJ, GroEL (MopA), and GroES (MopB). In the course of this work, we confirmed that the catalytic subunit of the ATP-dependent Clp protease (also known as Ti protease), ClpP, is derived from a larger precursor protein. Possible assignments of some of the hsl genes to known proteins are discussed.
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PMID:Characterization of twenty-six new heat shock genes of Escherichia coli. 834 64


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