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 epitopes have been localized for a set of monoclonal antibodies specific for the alpha subunit of the Escherichia coli RNA polymerase. The antibodies are classified into three groups based on their epitopic assignments. Group 1, mAb 123C2, maps in the N terminus of alpha between amino acids 1 and 23; Group 2 antibodies (mAb 129C4, mAb 124D1 and mAb 121C5) map in the central region between amino acids 190 and 210; Group 3 antibodies (mAb 130B1 and mAb 125C6) map in the C terminus between amino acids 310 and 320. mAb 130C2 is anomalous since it maps to the N terminus between amino acids 1 and 23 as well as to the C terminus between amino acids 320 and 329. The antibodies were used to investigate the role of alpha in transcription activation with cAMP receptor protein-dependent promoters. Three antibodies (130C2, 121C5, and 125C6) inhibited cAMP receptor protein-dependent initiation with lac P+ but not with lac UV5 or gal P+. Inhibition was observed with free RNA polymerase and the closed promoter complex; the preformed open promoter complex was insensitive. Only lac P+ was sensitive to these anti-alpha antibodies supporting the concept that the mode of interaction of RNA polymerase with cAMP receptor protein differs between lac P+ and gal P+.
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PMID:Epitope mapping and functional characterization of monoclonal antibodies specific for the alpha subunit of Escherichia coli RNA polymerase. 752 31

Previous studies with U937 cells, a human monocyte cell line, have shown that the activity of cyclic nucleotide phosphodiesterase 4 (PDE4) is increased by agents that elevate cyclic AMP content. The present experiments were conducted to determine 1) whether an increase in PDE4 steady-state message and/or protein accompanies the up-regulation of PDE4 activity and 2) whether the up-regulation changes the functional responses of U937 cells to activators of adenylyl cyclase. To up-regulate PDE4 activity, U937 cells were treated for 4 h with a combination of 1 microM salbutamol, a beta-adrenoceptor agonist, and 30 microM rolipram, a PDE4 inhibitor. Cells were washed extensively to remove drugs and used immediately in various experimental protocols. Reverse transcriptase-polymerase chain reactions conducted with primers specific for the four PDE4 subtypes suggested that pretreatment with salbutamol and rolipram increased steady-state mRNA levels of PDE4A and PDE4B, but not PDE4C or PDE4D. Immunoblot analyses using two rabbit polyclonal antibodies, one directed against human recombinant PDE4A and PDE4D and a second directed against human recombinant PDE4B, revealed bands of immunoreactivity corresponding to approximately 125 kDa (PDE4A) and approximately 70 kDa (PDE4B), respectively, that increased in intensity after cells were treated with salbutamol and rolipram. As demonstrated in both time course and concentration-response studies with prostaglandin E2 (PGE2), an agent that activates adenylyl cyclase by a non-beta-adrenoceptor-mediated mechanism, cAMP accumulation was substantially decreased in cells in which PDE4 activity had been up-regulated. The difference in PGE2-stimulated cAMP accumulation between control and PDE4 up-regulated cells was greatly reduced in the presence of rolipram, consistent with the notion that an increase in PDE4 activity was responsible for the heterologous desensitization. Functionally, up-regulation of PDE4 markedly decreased the ability of PGE2 to inhibit LTD4-induced Ca2+ mobilization in intact cells. A hypothetical implication of these results is that increasing PDE4 activity in vivo by administering beta-adrenoceptor agonists could exacerbate inflammatory processes by decreasing the activity of endogenous anti-inflammatory agents such as PGE2.
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PMID:Salbutamol up-regulates PDE4 activity and induces a heterologous desensitization of U937 cells to prostaglandin E2. Implications for the therapeutic use of beta-adrenoceptor agonists. 755 25

Studies were performed to determine the mechanism underlying deficient arginine vasopressin (AVP)-stimulated adenylyl cyclase activity in chronic renal failure (CRF). As compared to control, principal cells cultured from the inner medullary collecting tubule of rats previously made uremic by 5/6 nephrectomy fail to accumulate cAMP when stimulated with AVP. CRF cells do respond normally to forskolin or cholera toxin and the defect in AVP responsiveness is not prevented by treatment with pertussis toxin, by cyclooxygenase inhibition, or by inhibition or down-regulation of protein kinase C. In contrast to their lack of responsiveness to AVP, CRF cells respond normally to other agonists of adenylyl cyclase such as isoproterenol or prostaglandin E2. Plasma membranes prepared from the inner medullae of CRF rats exhibit a marked decrease in apparent AVP receptor number but no change in the apparent number of beta adrenergic receptors. Reverse transcriptase PCR of total RNA from the inner medullae of CRF animals reveals virtual absence of V2 receptor mRNA; mRNA for alpha s is present in normal abundance. These studies indicate that AVP resistance in CRF is due, at least in part, to selective down-regulation of the V2 receptor as a consequence of decreased V2 receptor mRNA.
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PMID:Vasopressin resistance in chronic renal failure. Evidence for the role of decreased V2 receptor mRNA. 761 8

There is increasing evidence that parathyroid hormone (PTH) and PTH-related peptides (PTHrP) are involved in normal skin cell growth; therefore, we investigated whether the PTH/PTHrP receptor was expressed in cultured human keratinocytes and dermal fibroblasts. Northern analyses of poly (A)+ RNA isolated from cultured fibroblasts revealed two PTH/PTHrP receptor transcripts with one major band at 2.5 kb and one minor band at 2.3 kb. These transcripts were consistent with those found in human osteosarcoma cells, which are known to express PTH/PTHrP-R mRNAs. In contrast, after repeated Northern analyses no PTH/PTHrP receptor transcripts were found in poly (A)+ RNA isolated from cultured keratinocytes. Reverse-transcriptase/nested polymerase chain reaction analyses of total RNA isolated from cultured keratinocytes and fibroblasts confirmed the Northern analyses data that the PTH/PTHrP receptor was expressed in cultured fibroblasts but not in cultured keratinocytes. When cultured fibroblasts and keratinocytes were exposed to 10(-7) M PTH (1-34) there was a twofold increase in cAMP levels in the fibroblasts and no demonstrable increase was noted in keratinocytes. These results suggest that skin fibroblasts possess the classical PTH/PTHrP receptor and are target cells for PTH and PTHrP whereas keratinocytes do not have the receptor and are unresponsive to its N-terminal agonist in the stimulation of cAMP formation.
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PMID:Cultured human fibroblasts and not cultured human keratinocytes express a PTH/PTHrP receptor mRNA. 761 67

Using a functioning rat thyroid cell line (FRTL-5), we studied the effects of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) on thyroidal type I iodothyronine 5'-deiodination (I-5'-deiodination) and on the expression of I-5'-deiodinase (I-5'-D) mRNA. After 24 h incubation in medium containing 0.5 microM rT3 with a tracer amount of [125I]rT3, radioactivity of released 125I- was counted. Deiodination in live FRTL-5 cells was enhanced about three times from the basal level by the addition of TSH and was inhibited markedly by propylthiouracil and dose dependently by T4. These results suggest the suitability of this model for investigating I-5'-deiodination in live thyroid tissue. Basal and TSH-induced I-5'-deiodination were significantly inhibited by 100 ng/liter of IL-1 beta and IL-6, and the inhibitory effect of TNF-alpha was seen over 1 microgram/liter. I-5'-deiodination was restored by removal of the cytokines. TSH-induced cAMP production and (Bu)2cAMP-induced I-5'-deiodination were also inhibited by the cytokines. Catalase, dexamethasone, and indomethacin did not abolish the inhibitory effects of the cytokines. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed a marked suppression of I-5'-D mRNA expression by IL-1 beta and IL-6. We conclude that these cytokines inhibit the thyroidal type I I-5'-deiodination in the order of potency IL-1 beta > IL-6 >> TNF-alpha, probably by decreasing the I-5'-D mRNA level.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 on type I iodothyronine 5'-deiodination in rat thyroid cell line, FRTL-5. 762 12

The loop at the 52-position of the cAMP receptor protein (CRP) has been suggested as a potential site for contacting RNA polymerase on Class II promoters where the CRP binding site is located at position -41.5 (Bell, A., Gaston, K., Williams, R., Chapman, K., Kolb, A., Buc, H., Minchin, S., Williams, J., and Busby, S. (1990) Nucleic Acids Res. 18, 7243-7250). Using protein-protein photo-cross-linking, evidence is presented showing that the 52-loop of CRP is in physical proximity to the delta subunit of RNA polymerase holoenzyme. This interaction required the presence of a functional preinitiation complex. The CRP suppressor mutation, K52N, increased the efficiency of cross-linking, indicating an improved physical interaction between the CRP 52-loop and the delta subunit. Evidence for direct interaction between the CRP 156-162 loop and delta subunit of RNA polymerase on both gal and lac promoters are also provided. The data indicate that CRP bound to the gal promoter contacts both the alpha and delta 70 subunits of RNA polymerase.
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PMID:Evidence for contact between the cyclic AMP receptor protein and the delta 70 subunit of Escherichia coli RNA polymerase. 764 91

Transcription of the genes belonging to the phosphate (pho) regulon in Escherichia coli requires the specific activator protein PhoB, in addition to RNA polymerase containing the major sigma factor, sigma 70, which is encoded by rpoD. We previously isolated two mutant sigma 70s (D570G and E575K) that were specifically defective in transcribing the pho genes. The mutated sites were located near and within the first helix of the helix-turn-helix (HTH) motif or region 4.2 of sigma 70. To study further the role of the first helix of the HTH motif of sigma 70 in transcriptional activation by PhoB, we made a series of rpoD mutations that alter the motif and purified the mutant sigma 70 proteins. RNA polymerases containing the mutant sigma 70s Y571A, T572L, V576T, K578E and F580V showed reduced in vitro transcription from the pstS promoter, a representative pho promoter, in the presence of PhoB, whereas RNA polymerase containing another mutant sigma 70 (E574K) showed enhanced transcription from the promoter. Transcription from the activator-independent tac promoter and the pBR-P4 promoter, which is independent of PhoB and requires cAMP-CRP (cAMP receptor protein) for transcription, was affected at most only marginally by these sigma 70 mutations. These results provide further evidence that the first helix plays an important role in the specific interaction between RNA polymerase and PhoB protein bound to the pho promoters in transcriptional activation.
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PMID:Mutational analysis of the role of the first helix of region 4.2 of the sigma 70 subunit of Escherichia coli RNA polymerase in transcriptional activation by activator protein PhoB. 765 20

We showed that transcription of mitochondrial (mt) genes in Saccharomyces cerevisiae is governed in part by cellular cAMP levels, and that such transcriptional control is mediated via cAMP-dependent protein kinase (cAPK) activity. Here we use in vitro protein kinase assays with intact mitochondria from respiring cells to define protein substrates for mt cAPK. Our data show that there are at least eight mt proteins phosphorylated in a cAMP-dependent manner, ranging in M(r) from 96000 to 9500. Similar assays with organelles from an mtf1 mutant and its wild-type parent strain show no loss of any mt cAPK target proteins, suggesting that Mtflp (M(r) = 40000), the mt RNA polymerase specificity factor, does not require phosphorylation for activity. We further show, using double mutants for TPK1, TPK2, and TPK3, which encode catalytic subunits of the mt cAPK, that each of the eight mt substrate proteins is not phosphorylated equivalently by the individual catalytic subunits.
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PMID:Substrates for yeast mitochondrial cAMP-dependent protein kinase activity. 766 38

Activation of gene expression in eukaryotes generally involves the action of multiple transcription factors that function synergistically when bound near a particular target gene. Such effects have been suggested to occur because multiple activators can interact simultaneously with one or more components of the basal transcription machinery. In prokaryotes, examples of synergistic effects on transcription are much more limited and can often be explained by cooperative DNA binding. Here we show that the Escherichia coli cAMP receptor protein (CRP) functions synergistically to activate transcription from a derivative of the lac promoter that bears a second CRP-binding site upstream of the natural binding site. We present evidence indicating that cooperative DNA binding of two CRP dimers does not account for the magnitude of the observed cooperative activation. We suggest, instead, that the two dimers stimulate transcription directly by contacting two distinct surfaces of RNA polymerase simultaneously. Thus, synergistic activation by CRP may provide a relatively simple model for examining the molecular basis of such effects in higher organisms.
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PMID:Synergistic activation of transcription by Escherichia coli cAMP receptor protein. 768 95

Salivary-specific and cAMP-inducible expression of the rat proline-rich protein gene RP4 is dependent on a 28-base pair sequence of a salivary-specific cAMP response element (SCRE) (Lin, H. H., and Ann, D. K. (1992) Gene Expression 2, 365-377). To unravel its trans-acting factor(s), we used double-stranded oligoprobes corresponding to the SCRE to screen a randomly primed lambda gt11 cDNA expression library made from RNA of rat salivary cells. In this report, we describe the cDNA cloning of these helix-loop-helix SCRE-binding proteins (SCBPs) and demonstrate that there are at least three isoforms in salivary cells, namely SCBP alpha, SCBP beta, and SCBP gamma. RNA polymerase chain reaction and sequence analyses further confirmed the existence of these three different SCBP isoforms, which code for putative proteins of 707, 706, and 682 amino acids, respectively. Expression of the cloned SCBP cDNAs in salivary cells stimulates the expression of a cotransfected reporter construct containing multicopies of the SCRE cloned upstream of the thymidine kinase promoter and the chloramphenicol acetyltransferase structural gene. This stimulation is much more pronounced in transfections in which SCBP alpha and SCBP beta are cotransfected than when they are transfected individually. Furthermore, when low concentrations of SCBP alpha and SCBP beta are cotransfected with the SCRE reporter gene, coexpression of the catalytic subunit of protein kinase A is required to efficiently activate the expression of the reporter gene. These results strongly suggest that the observed stimulation of the SCRE is achieved through the coordinated expression of the SCBP alpha, SCBP beta, and protein kinase A activities, perhaps via a direct association of the two SCBPs and their phosphorylation by protein kinase A. We conclude that the isolated SCBP alpha and SCBP beta cDNAs encode transcription activators that participate in the control of the inducible RP4 gene expression in salivary cells.
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PMID:The helix-loop-helix proteins (salivary-specific cAMP response element-binding proteins) can modulate cAMP-inducible RP4 gene expression in salivary cells. 768 70


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