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)

We previously described the purification and characterization of E1BF, a rat rRNA gene core promoter-binding factor that consists of two polypeptides of 89 and 79 kDa. When this factor was incubated in the absence of any exogenous protein kinase under conditions optimal for protein phosphorylation, the 79-kDa polypeptide of E1BF was selectively phosphorylated. The labeled phosphate could be removed from the E1BF polypeptide by treatment with calf intestinal alkaline phosphatase or potato acid phosphatase. Elution of the protein from the E1BF-promoter complex formed in an electrophoretic mobility-shift assay followed by incubation of the concentrated eluent with [gamma-32P] ATP resulted in the selective labeling of the 79-kDa band. The E1BF-associated protein kinase did not phosphorylate casein or histone H1. Fraction DE-B, a preparation containing RNA polymerase I and all polymerase I transcription factors (including E1BF), lost polymerase I transcriptional activity when treated with phosphatase. The phosphatase-induced inactivation of polymerase I activity associated with fraction DE-B could be reversed by the addition of purified E1BF. Treatment of purified E1BF with heat, SDS, or an ATP affinity analog eliminated its capacity to reactivate dephosphorylated fraction DE-B. These data demonstrate that (i) polymerase I promoter-binding factor E1BF contains an intrinsic substrate-specific protein kinase and (ii) E1BF is an essential polymerase I transcription factor that can modulate rRNA gene transcription by protein phosphorylation. Further, these studies have provided a direct means to identify a protein kinase or any other enzyme that can interact with a specific DNA sequence.
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PMID:E1BF is an essential RNA polymerase I transcription factor with an intrinsic protein kinase activity that can modulate rRNA gene transcription. 192 88

The two forms of RNA polymerase II that exist in vivo, phosphorylated (IIO) and nonphosphorylated (IIA), were purified to apparent homogeneity from HeLa cells. The nonphosphorylated form preferentially binds to the preinitiation complex. RNA polymerase II in the complex was converted by a cellular protein kinase to the phosphorylated form.
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PMID:The nonphosphorylated form of RNA polymerase II preferentially associates with the preinitiation complex. 194 17

The relationship between NS protein phosphorylation and RNA polymerase activities was determined in nucleocapsids purified from vesicular stomatitis virus grown in BHK cells. Phosphate incorporation into endogenous NS protein under transcription conditions reached a maximum value of 0.06 mol/mol of NS within 20 to 30 min, while RNA synthesis remained linear for 90 min. Phosphate incorporation into NS increased further upon addition of kinase-free NS protein but not upon addition of nucleocapsid kinase (prepared as described below), indicating that cessation of NS phosphorylation under transcribing conditions was due to substrate exhaustion. When NS was phosphorylated with 32P, less than 8% of the radiolabel was lost during subsequent transcription, indicating that this phosphate did not turn over. Treatment of nucleocapsids with 5'-p-fluorosulfonylbenzoyl adenosine resulted in greater than 90% inhibition of NS phosphorylation but had no effect on RNA polymerase activity. Fast protein liquid (Superose-6) chromatography of a nucleocapsid (L + NS) fraction resulted in complete separation of the viral (L + NS) protein from NS-phosphorylating activity. The addition of this kinase-free (L + NS) fraction to a kinase-deficient N-RNA fraction reconstituted an active RNA polymerase containing less than 20% of the original NS-phosphorylating activity. These results demonstrate that NS-phosphorylating activity is unnecessary during vesicular stomatitis virus RNA synthesis and indicate that all of the protein kinase(s) present in purified nucleocapsids is probably of cellular rather than viral origin.
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PMID:Phosphorylation of NS protein by vesicular stomatitis virus nucleocapsids: lack of effect during RNA synthesis and separation of kinase from L protein. 216 40

RNA polymerase 1 activity and nucleolar volume have been reported to increase in hepatocytes from rats fed a protein-free diet. Phosphorylation in vitro of a 110-kDa protein was enhanced in nuclei and nucleoli from livers of rats fed a protein-free diet. In nuclear extracts the 110-kDa protein in heat-treated nuclei was much more phosphorylated than from control liver. In contrast, casein kinase activity in the nuclear extract from control liver was comparable to that from livers of rats fed a protein-free diet. Nuclear extracts from control rat liver and livers of rats fed a protein-free diet were fractionated by DEAE-cellulose column chromatography. Casein kinase II (NII) eluted at around 0.17 M NaCl scarcely phosphorylates the 110-kDa protein. Chromatography of the nuclear extract from livers of rats fed a protein-free diet, but not from control liver, yielded fractions which eluted at 0.21-0.25 M NaCl and predominantly phosphorylated the 110-kDa protein. The phosphorylation of 110-kDa protein was not appreciably affected by a heparin concentration of 5 micrograms/ml, which completely inhibited casein kinase II. In addition, phosphorylation of the 110-kDa protein in liver nucleoli from rats fed a protein-free diet showed a lower sensitivity to heparin than that in control rat liver nucleoli. These results suggest that enhanced phosphorylation of the nuclear 110-kDa protein in livers from rats fed a protein-free diet is due to the induction of a 110-kDa protein kinase distinct from casein kinase II.
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PMID:Enhanced phosphorylation of a nucleolar 110-kDa protein in rat liver by dietary manipulation. 216 18

Changes in protein kinase activities are thought to contribute to the alteration of gene expression after heat shock and related stresses. In an attempt to identify enzymes which might be involved in both chromatin structure modification and transcriptional switch in heat-shocked cells, we have studied protein kinase activities in heat-shocked cell lysates with two exogenous substrates: a tetramer of a heptapeptide (heptapeptide 4) corresponding to the RNA polymerase II C-terminal domain (CTD), and the histone H1. Heat-shock and arsenite stress were found to stimulate strongly CTD kinase activity. H1 kinase activity was also stimulated but more weakly. Stimulation of CTD and H1 kinases occurs mainly at the early phase of recovery and by a process which is independent of protein synthesis. The stress-induced H1 kinase is shown to contain a molecule related to the mitotic-promoting factor (MPF) Cdc2 component. On the other hand, though Cdc2-related protein has also been reported to be part of a CTD kinase complex, we show that the stress-induced CTD kinase activity corresponds to a distinct entity. It is proposed that stress activation of CTD kinase might be involved in changing the specificity of RNA polymerase II.
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PMID:Heat-shock and related stress enhance RNA polymerase II C-terminal-domain kinase activity in HeLa cell extracts. 217 28

Bacteriophage T7 expresses a serine/threonine-specific, cAMP-independent protein kinase activity encoded by the early gene 0.7. The phosphoproteins specifically resulting from gp0.7 protein kinase expression in T7-infected Escherichia coli have been examined by one-dimensional, SDS-polyacrylamide gel electrophoresis. Only seven major, stable phosphoproteins dependent on gp0.7 protein kinase expression are observed. Two of the gp0.7 protein kinase-specific phosphoproteins observed have been previously identified: the beta' subunit of RNA polymerase and the RNA processing enzyme RNase III. The gp0.7-catalyzed protein phosphorylation activity appears at 9-11 min postinfection at 30 degrees. The new phosphoproteins have a metabolic stability comparable to that of uninfected cell phosphoproteins. T7 protein kinase expression causes the phosphorylation of the same, limited set of proteins in B, C, or K strains of E. coli. Expression of the T3 and BA14 phage protein kinase activities also produces the same phosphoproteins.
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PMID:Protein kinase of bacteriophage T7 induces the phosphorylation of only a small number of proteins in the infected cell. 218 69

The gene of catalytic domain of the protein kinase of RSV-scr was cloned into the BamHI cloning site of translation vector pET-8c which containing T7 RNA polymerase promotor, and transformed BL21 (DE3) pLys S (Studier and Moffatt, 1986). The putative molecular weight of the protein was about 33 kd as evaluated on the basis of its nucleotide size showed the identical mobility in SDS-polyacrylamide gel electrophoresis. However, yield of protein production was not high, probably, because of its instability in Escherichia coli.
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PMID:Subcloning and trial of expression of the protein kinase catalytic domain of RSV-src gene. 221 79

Bacteriophage RNA polymerases are widely used to synthesize defined RNAs on a large scale in vitro. Unfortunately, the RNA product contains a small proportion of contaminating RNAs, including complementary species, which can lead to errors of interpretation. We cloned the gene encoding Ad2 VA RNAI into a vector containing a T7 RNA polymerase promoter in order to generate large quantities of VA RNA for the study of its interaction with the dsRNA-dependent protein kinase DAI. Exact copies of VA RNAI were synthesized efficiently, but were contaminated with small amounts of dsRNA which activated DAI and confounded interpretation of kinase assays. We therefore developed a method to remove the dsRNA contaminants, allowing VA RNAI and mutants to be tested for their ability to activate or inhibit DAI. This method appears to be generally applicable.
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PMID:Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNAI from a T7 vector. 221 12

Various agents were tested for their effects on microbial proteases, which activity was monitored by the analysis of cleaved peptide bands in SDS-polyacrylamide gel electrophoresis. Using casein as a substrate, fungal protease (type XIX) was inhibited by the phenyl methyl sulphonyl fluoride, chymostatin, antipain and leupeptin, while bacterial protease (type XXVI) was inhibited by phosphatidyl glycerol, phosphatidyl inositol and sphingosine. MS2 RNA exerted minor inhibition on the bacterial proteolysis of regulatory subunits of cyclic AMP-dependent protein kinase (A-PK). The cleavage of DNA binding protein by both proteases was inhibited, in the presence of MS2 RNA and lambda DNA. In comparison, phosphatidyl serine slightly stimulated the fungal protease on the cleavage of ribonuclease T1. RNA polymerase is a good substrate of the bacterial protease as indicated by the generation of multiple cleaved peptide fragments, whereas alkaline phosphatase is not susceptible to proteolysis.
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PMID:A further study on the regulation of microbial proteases. 222 36

The largest subunit of mammalian RNA polymerase II contains at its C terminus an unusual domain consisting of multiple tandem repeats of the seven-amino acid consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This domain is unphosphorylated in RNA polymerase IIA and extensively phosphorylated in RNA polymerase IIO. To investigate the role of the C-terminal domain and the functional significance of its phosphorylation, changes in the level of phosphorylation were followed as a function of the position of RNA polymerase II in the transcription cycle. Complexes were formed with 32P-labeled RNA polymerase IIA and separated from the free polymerase by gel filtration. The phosphorylation state of the RNA polymerase II largest subunit was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results indicate that RNA polymerase IIA interacts with the template-committed complex to form a stable preinitiation complex. RNA polymerase IIA associated with such complexes is converted to RNA polymerase IIO in the presence of ATP prior to the formation of the first phosphodiester bond. Furthermore, the observation that purified preinitiation complexes can catalyze the conversion of RNA polymerase IIA to IIO indicates that the protein kinase(s) responsible for phosphorylation of the C-terminal domain is a component of such complexes. The concentration of ATP required for the phosphorylation of RNA polymerase II associated with the preinitiation complex is two to three orders of magnitude lower than that required for the conversion of RNA polymerase IIA to IIO free in solution. These results support the idea that phosphorylation of the C-terminal domain of RNA polymerase subunit IIa occurs subsequent to the association of enzyme with the promoter and prior to the initiation of transcription.
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PMID:Phosphorylation of RNA polymerase IIA occurs subsequent to interaction with the promoter and before the initiation of transcription. 237 91


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