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 RNA-dependent RNA polymerase of influenza virus A/PR/8 was isolated from virus particles by stepwise centrifugation in cesium salts. First, RNP (viral RNA-NP-P proteins) complexes were isolated by glycerol gradient centrifugation of detergent-treated viruses and subsequently NP was dissociated from RNP by cesium chloride gradient centrifugation. The P-RNA (P proteins-viral RNA) complexes were further dissociated into P proteins and viral RNA by cesium trifluoroacetate (CsTFA) gradient centrifugation. The nature of P proteins was further analyzed by glycerol gradient centrifugation and immunoblotting using monospecific antibodies against each P protein. The three P proteins, PB1, PB2, and PA, sedimented altogether as fast as the marker protein with the molecular weight of about 250,000 Da. Upon addition of the template vRNA, the RNA-free P protein complex exhibited the activities of capped RNA cleavage and limited RNA synthesis. When a cell line stably expressing cDNAs for three P proteins and NP protein was examined, the three P proteins were found to be co-precipitated by antibodies against the individual P proteins. These results indicate that the influenza virus RNA-dependent RNA polymerase is a heterocomplex composed of one each of the three P proteins and that the RNA-free RNA polymerase can be isolated in an active form from virus particles. Furthermore, the three P proteins form a complex in the absence of vRNA.
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PMID:Purification and molecular structure of RNA polymerase from influenza virus A/PR8. 235 36

We have identified a mitochondrial protein from Saccharomyces cerevisiae which confers the ability to recognize mitochondrial promoters onto a nonspecifically transcribing mitochondrial core RNA polymerase and we have purified this specificity factor 10,700-fold from a whole cell extract. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fraction followed by elution and renaturation of protein activity shows that the specificity factor is a 43-kDa polypeptide which directs mitochondrial core RNA polymerase to promoters belonging to rRNA-, tRNA-, and protein-encoding genes, as well as to mitochondrial replication origins. Gel filtration and glycerol gradient sedimentation studies indicate that the specificity factor shows little association with core RNA polymerase in the absence of DNA, and that it behaves like a monomeric 43-kDa protein.
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PMID:Specificity factor of yeast mitochondrial RNA polymerase. Purification and interaction with core RNA polymerase. 244 67

The properties of the two monoclonal antibodies which were found to inhibit cyclic AMP receptor protein (CRP)-stimulated abortive initiation without affecting cAMP binding (Li, X.-M., and Krakow, J. S. (1986) J. Biol. Chem. 260, 4378-4383) have been characterized. Binding of monoclonal antibody (mAb) 66C3 to CRP is stimulated by cAMP while CRP binding by mAb 63B2 is not affected by cAMP. Binding of cAMP-CRP-mAb 63B2 to the lac P+ DNA is completely inhibited. Whereas cAMP-CRP forms a stable complex only at the CRP site 1 of the lac P+ promoter fragment, cAMP-CRP-mAb 66C3 binds to both site 1 and site 2. DNase I footprinting using a HpaII fragment carrying only the lac site 2 does not show any protection by cAMP-CRP-mAb 66C3. With the lac L8UV5 promoter, binding is not seen at either the L8 site 1 or the unaltered site 2. In the presence of 25% glycerol, cAMP-CRP-mAb 66C3 binds to both L8 site 1 and site 2. RNA polymerase is unable to bind to the cAMP-CRP-mAb 66C3-lac P+ complex. In the presence of RNA polymerase, cAMP-CRP forms a stable complex at the L8 site 1, the subsequent addition of mAb 66C3 results in the release of CRP. The CRP present in the lac P+ open promoter complex is partially resistant to subsequent incubation with mAb 66C3. The results provide further evidence regarding possible contacts between CRP and RNA polymerase involved in establishing the open promoter complex.
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PMID:Monoclonal antibodies that inhibit activation of transcription by the Escherichia coli cyclic AMP receptor protein. 244 41

Myxococcus xanthus, a myxobacterium, contains a peculiar branched RNA-linked DNA called msDNA. Reverse transcriptase has been shown to be required for the production of msDNA. Existence of proteins that bind to one of the two msDNAs in M. xanthus, msDNA.Mx162, was examined by gel retardation assays. Total cell-free extract yielded two distinct retarded bands. Both bands were sensitive to treatment with proteinase K, indicating that there is a protein(s) that is able to bind to msDNA. Further, the formation of the bands was inhibited by the addition of nonradioactive msDNA but not by a large excess of poly(dA) in the presence of a 5000-fold excess of poly(dI.dC).poly(dI.dC). In vivo footprinting using dimethyl sulfate revealed that the deoxynucleotide stretch from 60 to 161 is protected. When a M. xanthus cell lysate was centrifuged in a 16-30% glycerol gradient, msDNA was found to sediment in two peaks: a major peak corresponding in size to 14 S, and a minor one at 5 S. These results indicate that msDNA.Mx162 exists as a complex with specific proteins in the cell.
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PMID:Myxococcus xanthus msDNA.Mx162 exists as a complex with proteins. 250 5

The rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (ATP:D-fructose-6-phosphate 2-phosphotransferase/D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC 2.7.1.105/EC 3.1.3.46) and its separate kinase domain were expressed in Escherichia coli by using an expression system based on bacteriophage T7 RNA polymerase. The bifunctional enzyme (470 residues per subunit) was efficiently expressed as a protein that starts with the initiator methionine residue and ends at the carboxyl-terminal tyrosine residue. The expressed protein was purified to homogeneity by anion exchange and Blue Sepharose chromatography and had kinetic and physical properties similar to the purified rat liver enzyme, including its behavior as a dimer during gel filtration, activation of the kinase by phosphate and inhibition by alpha-glycerol phosphate, and mediation of the bisphosphatase reaction by a phosphoenzyme intermediate. The expressed 6-phosphofructo-2-kinase also started with the initiator methionine but ended at residue 257. The partially purified kinase domain was catalytically active, had reduced affinities for ATP and fructose 6-phosphate compared with the kinase of the bifunctional enzyme, and had no fructose-2,6-bisphosphatase activity. The kinase domain also behaved as an oligomeric protein during gel filtration. The expression of an active kinase domain and the previous demonstration of an actively expressed bisphosphatase domain provide strong support for the hypothesis that the hepatic enzyme consists of two independent catalytic domains encoded by a fused gene.
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PMID:Expression of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its kinase domain in Escherichia coli. 255 38

Using isolated nuclei prepared from influenza virus-infected HeLa cells, factors affecting the synthesis of two species of positive-sense RNA transcripts, i.e., mRNA and cRNA (complementary RNA to vRNA) were analyzed. In the presence of low concentrations of salt, both mRNA and cRNA were synthesized, whereas in the presence of high concentrations of salt, mRNA was synthesized predominantly. Salt-extracts of nuclei (NE) mainly produced cRNA while mRNA was a major product synthesized by salt-treated nuclei (delta N). In the presence of high concentrations of salt, the NE produced mRNA instead of cRNA. After centrifugation of the NE, the precipitates (NEP) predominantly produced mRNA while the supernatant (NES) alone exhibited a low level of cRNA synthesis activity. With the addition of the NES fraction, mRNA synthesis by the NEP was switched to cRNA synthesis. Glycerol gradient centrifugation of the NES fraction in the presence of high salt yielded vRNA-RNA polymerase complexes that catalyzed mRNA synthesis. These observations indicate that a regulatory factor(s) that can be dissociated from vRNA-RNA polymerase complexes upon exposure to high ionic strength is involved in the switch from mRNA to cRNA synthesis. This activity was not detected in nuclear extracts prepared from uninfected cells, suggesting that such a factor(s) is either encoded by the virus genome or induced by virus infection.
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PMID:In vitro synthesis of influenza viral RNA: biochemical complementation assay of factors required for influenza virus replication. 280 17

The in vitro transcription reaction of bluetongue virus (BTV) is characterized by a core-mediated, temperature-dependent inhibition at high core concentrations and temperatures (Van Dijk & Huismans, 1980; Huismans, Van Dijk & Els, 1987a). It has been found that this inhibition is reversible and that an inactivated transcriptase reaction mixture can be reactivated by lowering the temperature of the reaction from 37 degrees C to 28 degrees C. In the same way it is possible to inactivate a reaction by increasing the incubation temperature from 28 degrees C to 37 degrees C. It was also found that the inhibition is counteracted by the addition of sucrose or glycerol. At relatively low core concentrations and in the presence of sucrose it is possible to obtain conditions under which transcription at 37 degrees C is more efficient than at 28 degrees C. The latter conditions probably reflect much better the in vivo temperature optimum for the BTV transcriptase than the in vitro conditions at very high core concentrations.
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PMID:The identification of factors capable of reversing the core-mediated inhibition of the bluetongue virus transcriptase. 283 1

Two general transcription factors (IIE and IIB) (TF) were purified from HeLa cell nuclear extracts and shown to be absolutely required, along with two additional factors (IIA and IID) and RNA polymerase II, for specific transcription initiation at the adenovirus major late promoter. TFIIB and TFIIE were also required, in addition to TFIIA, TFIID, RNA polymerase II, and the adenovirus 2 major late promoter, for the formation of a (preinitiation) complex that could initiate transcription (upon addition of nucleoside triphosphates) in the presence of heparin concentrations which inhibited the action of unbound factors. Glycerol gradient analyses indicated independent interactions of TFIIE with TFIIB and with the purified RNA polymerase II, but not with RNA polymerase III. Transcription factors IIB and IIE were also shown to be required for specific initiation of transcription from several cellular and viral class II promoters.
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PMID:Factors involved in specific transcription by mammalian RNA polymerase II. Purification and functional analysis of initiation factors IIB and IIE. 302 9

A high-molecular-weight protein complex that is capable of accurate transcription initiation and termination of vaccinia virus early genes without additional factors was demonstrated. The complex was solubilized by disruption of purified virions, freed of DNA by passage through a DEAE-cellulose column, and isolated by glycerol gradient sedimentation. All detectable RNA polymerase activity was associated with the transcription complex, whereas the majority of enzymes released from virus cores including mRNA (nucleoside-2'-O)methyltransferase, poly(A) polymerase, topoisomerase, nucleoside triphosphate phosphohydrolase II, protein kinase, and single-strand DNase sedimented more slowly. Activities corresponding to two enzymes, mRNA guanylyltransferase (capping enzyme) and nucleoside triphosphate phosphohydrolase I (DNA-dependent ATPase), partially sedimented with the complex. Silver-stained polyacrylamide gels, immunoblots, and autoradiographs confirmed the presence of subunits of vaccinia virus RNA polymerase, mRNA guanylyltransferase, and nucleoside triphosphate phosphohydrolase I, as well as additional unidentified polypeptides, in fractions with transcriptase activity. A possible role for the DNA-dependent ATPase was suggested by studies with ATP analogs with gamma-S or nonhydrolyzable beta-gamma-phosphodiester bonds. These analogs were used by vaccinia virus RNA polymerase to nonspecifically transcribe single-stranded DNA templates but did not support accurate transcription of early genes by the complex. Transcription also was sensitive to high concentrations of novobiocin; however, this effect could be attributed to inhibition of RNA polymerase or ATPase activities rather than topoisomerase.
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PMID:Sedimentation of an RNA polymerase complex from vaccinia virus that specifically initiates and terminates transcription. 303 83

Immunoblotting of size-separated whole cell proteins permitted the study of protein-protein interaction. Briefly, proteins obtained from cleared cell lysates of Escherichia coli were separated by glycerol gradient centrifugation and analysed by blotting against a set of specific antibodies. We have applied this procedure to the assembly of 11 N-terminal amber fragments of the beta subunit of E. coli RNA polymerase ranging in size between 97% and 23% the length of the intact beta polypeptide (1342 amino acids). In this way, we have been able to define regions on the beta polypeptide involved in the assembly of RNA polymerase.
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PMID:Assembly of amber fragments of the beta subunit of Escherichia coli RNA polymerase. 304 46


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