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)

Chloroplast and etioplast RNA polymerase preparations each consist of a multi-subunit core and a set of three sigma-like transcription factors, SLF67, SLF52 and SLF29. Despite this structural similarity, the enzymes from either plastid type are functionally distinct, as is reflected by their different promoter usage and the tight core-SLF association in the etioplast but not the chloroplast holoenzyme. We tested whether these differences are related to phosphorylation. Treatment of the chloroplast enzyme with protein kinase converted it to an etioplast-type form and vice versa, treatment of the etioplast enzyme with phosphatase generated chloroplast-type properties. Although both the core enzyme and the SLF polypeptides were phosphorylation targets, only the SLFs seem to confer plastid-type-specific DNA binding characteristics. Methylation interference and DNase I footprint patterns in the psbA promoter region were found to correlate with the phosphorylation state of the chloroplast and etioplast enzymes.
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PMID:Phosphorylation and dephosphorylation affect functional characteristics of chloroplast and etioplast transcription systems from mustard (Sinapis alba L.). 849 Nov 68

Enhancer-dependent transcription in enteric bacteria depends upon an activator protein that binds DNA far upstream from the promoter and an alternative sigma factor (sigma 54) that binds with the core RNA polymerase at the promoter. In the photosynthetic bacterium Rhodobacter capsulatus, the NtrB and NtrC proteins (RcNtrB and RcNtrC) are putative members of a two-component system that is novel because the enhancer-binding RcNtrC protein activates transcription of sigma 54-independent promoters. To reconstitute this putative two-component system in vitro, the ReNtrB protein was overexpressed in Escherichia coli and purified as a maltose-binding protein fusion (MBP-RcNtrB). MBP-RcNtrB autophosphorylates in vitro to the same steady state level and with the same stability as the Salmonella typhimurium NtrB (StNtrB) protein but at a lower initial rate. MBP-RcNtrB autophosphorylates the S.typhimurium NtrC (St-NtrC) and RcNtrC proteins in vitro. The enteric NtrC protein is also phosphorylated in vivo by RcNtrB because plasmids that encode either RcNtrB or MBP-Rc-NtrB activate transcription of an NtrC-dependent nifL-lacZ fusion. The rate of phosphotransfer to RcNtrC and autophosphatase activity of phosphorylated RcNtrC (RcNtrC---P) are comparable to the StNtrC protein. However, the RcNtrC protein appears to be a specific RcNtrB P phosphatase since RcNtrC is not phosphorylated by small molecular weight phosphate compounds or by the StNtrB protein. RcNtrC forms a dimer in solution, and RcNtrC - P binds the upstream tandem binding sites of the g1nB promoter 4-fold better than the unphos-phorylated RcNtrC protein, presumably due to oligomerization of RcNtrC -P. Therefore, the R. capsulatus NtrB and NtrC proteins form a two-component system similar to other NtrC-like systems, where specific Rc- NtrB phosphotransfer to the RcNtrC protein results in increased oligoinerization at the enhancer but with subsequent activation of a sigma 54-independent promoter.
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PMID:In vitro reconstitution and characterization of the Rhodobacter capsulatus NtrB and NtrC two-component system. 862 57

Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two cell types that are formed by an asymmetric cell division. Major changes in the pattern of transcription during sporulation are brought about by the synthesis of new sigma factors (sigma), which are subunits of RNA polymerase that determine promoter specificity. Transcription in the smaller prespore cell type is initiated by a sigma factor called sigma F, the activity of which is subject to tight spatial and temporal control. It is negatively regulated by an anti-sigma factor, SpoIIAB, which is in turn controlled by an anti-anti-sigma factor, SpoIIAA. SpoIIAA and SpoIIAB participate in two contrasting reactions in vitro. In the presence of ATP, the proteins interact transiently and SpoIIAA is inactivated by phosphorylation on a specific serine residue; SpoIIAA then remains free to inhibit sigma F. In the presence of ADP, SpoIIAA binds tightly to SpoIIAB, and sigma F is set free. Release of sigma F activity in vivo might thus be effected by a prespore-specific reduction in the ATP/ADP ratio. Genetic experiments have implicated a fourth protein, called SpoIIE, in this system. It now appears that SpoIIE has two important and independent functions in the establishment of the prespore-specific transcription by sigma F. First it regulates sigma F activity, probably acting as a phosphatase to regenerate the active, non-phosphorylated form of SpoIIAA. Second it controls the formation of the septum that generates the prespore compartment. Combination of these two functions in a single polypeptide may provide a means of coupling gene expression with morphogenesis.
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PMID:Control of the cell-specificity of sigma F activity in Bacillus subtilis. 873 76

RNA polymerase (RNAP) II is subject to extensive phosphorylation on the heptapeptide repeats of the C-terminal domain (CTD) of the largest subunit. An activity that is required for the dephosphorylation of yeast RNAP II in vitro has been purified from a yeast whole cell extract by >30,000-fold. The yeast CTD phosphatase activity copurified with two bands with apparent molecular masses of 100 and 103 kDa. The properties of the yeast CTD phosphatase are similar to those of a previously characterized CTD phosphatase from HeLa cells. These properties include stimulation by the general transcription factor IIF (TFIIF), competitive inhibition by RNAP II, magnesium dependence, and resistance to okadaic acid. Both the HeLa and yeast CTD phosphatases are highly specific for their cognate polymerases. Neither phosphatase functions upon the polymerase molecule from the other species, even though the heptapeptide repeats of the CTDs in yeast RNAP II and mammalian RNAP II are essentially identical. The activity of the highly purified CTD phosphatase is stimulated >300-fold by a partially purified fraction of TFIIF. Recombinant TFIIF did not substitute for the TFIIF fraction, indicating that an additional factor present in the TFIIF fraction is required for CTD phosphatase activity. These results show that yeast contains a CTD phosphatase activity similar to that of mammalian cells that is likely composed of at least two components, one of which is 100 and/or 103 kDa.
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PMID:Purification and characterization of an RNA polymerase II phosphatase from yeast. 879 10

The general stress response of the bacterium Bacillus subtilis is governed by a signal transduction network that regulates activity of the sigma(B) transcription factor. We show that this network comprises two partner-switching modules, RsbX-RsbS-RsbT and RsbU-RsbV-RsbW, which contribute to regulating sigma(B). Each module consists of a phosphatase (X or U), an antagonist protein (S or V), and a switch protein/kinase (T or W). In the downstream module, the W anti-sigma factor is the primary regulator of sigma(B) activity. If the V antagonist is phosphorylated, the W switch protein binds and inhibits sigma(B). If V is unphosphorylated, it complexes W, freeing sigma(B) to interact with RNA polymerase and promote transcription. The phosphorylation state of V is controlled by opposing kinase (W) and phosphatase (U) activities. The U phosphatase is regulated by the upstream module. The T switch protein directly binds U, stimulating phosphatase activity. The T-U interaction is governed by the phosphorylation state of the S antagonist, controlled by opposing kinase (T) and phosphatase (X) activities. This partner-switching mechanism provides a general regulatory strategy in which linked modules sense and integrate multiple signals by protein-protein interaction.
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PMID:Opposing pairs of serine protein kinases and phosphatases transmit signals of environmental stress to activate a bacterial transcription factor. 882 86

Nuclear transcription is repressed when eukaryotic cells enter mitosis. Using Xenopus egg extracts shifted to the mitotic state with recombinant cyclin B1 protein, we have been able to reproduce mitotic repression of transcription in vitro. Active RNA polymerase III transcription is observed in interphase extracts in the absence of added cyclin, but is strongly repressed by the induction of cdc2/cyclin B (maturation/mitosis promoting factor, MPF) kinase activity in the mitotic extract. Studies with protein kinase inhibitors show that protein phosphorylation is required for repression. Add-back experiments indicate that repression of class III gene transcription is due to inactivation of the transcription factor TFIIIB. TFIIIB is composed of the TATA-box binding protein (TBP) and TBP-associated factors of 75 and 92 kDa. In the present study, we show that TBP and a polypeptide of 92 kDa are substrates of the mitotic kinase in highly purified TF- IIIB fractions. We also show that a phosphatase present in the Xenopus egg extract can reactivate transcription after repression by the mitotic kinases. This result suggests a mechanism for reactivation of transcription after exit from mitosis into the G1 phase of the cell cycle. As for pol III genes, purified cdc2/cyclin B kinase is sufficient to inhibit transcription by RNA polymerase II in a reconstituted transcription system containing the basal transcription factors and polymerase.
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PMID:Repression of RNA polymerase II and III transcription during M phase of the cell cycle. 898 11

Current research into cytokine production in tissue sections relies on the detection of cytokine proteins using a variety of immunohistochemical methods. The disadvantages of this technique are that precise localization to a particular cell is difficult and it is uncertain whether the cells detected by this method are the origin or target of the cytokine or rather have nonspecifically absorbed the secreted cytokine. This question can be clarified using in situ hybridization, but current techniques are insensitive, poorly localizing, or time consuming. Biotin-labeled riboprobes were generated from cDNA fragments sandwiched between two RNA polymerase promoters (SP6 and T7 RNA polymerases) using a commercial riboprobe generation kit containing biotin-labeled UTP. The in situ hybridization technique was used to demonstrate cytokine mRNA in a range of tissues containing an inflammatory infiltrate and with a range of cytokine probes. This technique of in situ hybridization was combined with immunohistochemistry using an immunoalkaline phosphatase technique to show the powerful combination of these two techniques. The biotin-labeled riboprobes were sensitive enough to detect a range of cytokine mRNAs in a variety of tissue sections. The technique can be completed over a 24-h period and produces a stable color product that can be stored for long periods and can be quantitated using image analysis techniques. This technique was performed on paraffin-embedded tissue as well as cryosections and allowed for the detection of mRNA in archival tissue. It was also successfully combined with immunohistochemical techniques to determine simultaneously the localization of a cytokine product in particular cell lineages. A nonradioactive method for in situ hybridization using biotin-labeled riboprobes is described; it is capable of detecting mRNA products from a range of genes in a variety of tissue samples. An amplification step in the method enhances the sensitivity to a level that approaches that of radioactive methods, while maintaining the speed, safety, and simplicity of an immunoperoxidase detection system. The ability to use paraffin-embedded tissue with this method allows for improved tissue architecture and examination of archival tissue. These features should ensure greater use of in situ hybridization techniques in future research studies.
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PMID:A nonradioactive method of in situ hybridization that uses riboprobes and paraffin-embedded tissue and its combination with immunohistochemistry. 902 35

Cytidine 5'-triphosphate (CTP):phosphocholine cytidylyltransferase (EC 2.7.7.15) catalyses the synthesis of the active metabolic intermediate cytidine diphosphocholine (which is mainly used in the synthesis of choline-containing phospholipids). It is a rate-limiting reaction in choline phospholipid biosynthesis in many cells. In this study, a microassay is reported for the detection of this enzyme in small numbers of cells. This enzyme was present in mouse oocytes and at all stages during preimplantation development. Enzyme activity was destroyed by boiling but increased with time and number of embryos in the reaction. Activity in two-cell embryos was dependent on Mg2+ but independent of Ca2+ and was enhanced by the addition of 1 microgram lysophosphatidylethanolamine ml-1 to the reaction mixture. Activity was apparently dependent upon the phosphorylation status of the enzyme since the absence of the phosphatase inhibitor NaF caused a significant inhibition of activity. The enzyme in oocytes had a specific activity of 2.8 +/- 0.3 fmol cytidine diphosphocholine (CDP-choline) per oocyte min-1 (mean +/- SEM). The specific activity in two-cell and eight-cell embryos and blastocysts was not different from that of oocytes. Fertilized one-cell embryos had significantly less activity (1.4 +/- 0.05 fmol CDP-choline produced per embryo min-1) than other stages studied. Furthermore, the enzyme present in one-cell embryos was not capable of being further activated by the addition of exogenous lysophosphatidylethanolamine to the reaction. The increase in activity from the one-cell to the two-cell stage was not inhibited by alpha-amanitin (an inhibitor of RNA polymerase II), cycloheximide (a protein synthesis inhibitor) [1-(5-isoquinolinesulfonyl)-2-methylpiperazine, HCl]dihydrochloride (H-7; a protein kinase inhibitor) and was independent of cell-cycle progression; these results suggest that enzyme activity is independent of transcription, protein synthesis and the action of some kinases, including cell-cycle-dependent kinases. This study provides the first description of cytidylyltransferase in the early mammalian embryo.
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PMID:CTP:phosphocholine cytidylyltransferase activity in the preimplantation mouse embryo. 930 73

We previously identified ZNF74 as a developmentally expressed gene commonly deleted in DiGeorge syndrome. ZNF74 encodes an RNA-binding protein tightly associated with the nuclear matrix and belongs to a large subfamily of Cys2-His2 zinc finger proteins containing a KRAB (Kruppel-associated box) repressor motif. We now report on the multifunctionality of the zinc finger domain of ZNF74. This nucleic acid binding domain is shown here to function as a nuclear matrix targeting sequence and to be involved in protein-protein interaction. By far-Western analysis and coimmunoprecipitation studies, we demonstrate that ZNF74 interacts, via its zinc finger domain, with the hyperphosphorylated largest subunit of RNA polymerase II (pol IIo) but not with the hypophosphorylated form. The importance of the phosphorylation in this interaction is supported by the observation that phosphatase treatment inhibits ZNF74 binding. Double immunofluorescence experiments indicate that ZNF74 colocalizes with the pol IIo and the SC35 splicing factor in irregularly shaped subnuclear domains. Thus, ZNF74 sublocalization in nuclear domains enriched in pre-mRNA maturating factors, its RNA binding activity, and its direct phosphodependent interaction with the pol IIo, a form of the RNA polymerase functionally associated with pre- mRNA processing, suggest a role for this member of the KRAB multifinger protein family in RNA processing.
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PMID:Direct interaction of the KRAB/Cys2-His2 zinc finger protein ZNF74 with a hyperphosphorylated form of the RNA polymerase II largest subunit. 934 35

We analyzed complexed and free prostate-specific antigen (PSA), the free/total PSA and complexed/free PSA ratios, acid phosphatase, and prostatic phosphatase in serum from 36 patients with prostatic carcinoma and from 48 non-neoplastic control patients (20 with prostatitis and 28 with benign prostatic hyperplasia). Receiver-operating characteristic plots showed that serum PSA was the most efficient variable, singly used, in discriminating neoplastic from non-neoplastic patients. At a cut-off value of 10.0 ng/ml, serum PSA had a diagnostic sensitivity of 87% and a diagnostic specificity of 83%. In particular, three patients with prostatic carcinoma and twenty non-neoplastic controls had serum PSA levels of between 4 and 10 ng/ml. The subsequent analysis of the serum free/total PSA ratio, in this subgroup, using a cut-off level of 15%, allowed us to classify correctly all prostatic cancer cases and 18/20 non-neoplastic diseases. We next analyzed PSA mRNA in circulating cells using an improved reverse-transcriptase polymerase chain reaction dot blot procedure, from six patients with prostatic carcinoma with distant metastases, and in seventeen with localized cancer. The analysis had a high sensitivity (up to dilutions 1:10(6) of total RNA from prostatic cancer cells vs total RNA from normal blood cells). The analysis revealed circulating micrometastatic cells in 3/6 (50%) cases of metastatic cancer and in 4/17 cases of localized cancer. To conclude, serum total PSA combined with the free/total PSA ratio is a very efficient algorithm in discriminating neoplastic from non-neoplastic prostatic diseases, while other mRNA species must be analyzed, in addition to PSA mRNA, in circulating cells to increase the efficiency in detecting metastatic prostatic cancer.
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PMID:Prostate-specific antigen (protein and mRNA) analysis in the differential diagnosis and staging of prostate cancer. 935 30


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