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)

Human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is 96% identical to the rat and bovine liver enzymes, and all of the critical catalytic and substrate binding residues in both the kinase and bisphosphatase domains are conserved in the three enzymes. However, in contrast to rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, which is readily expressed in an Escherichia coli T-7 RNA polymerase-based expression system, the human liver bifunctional enzyme could not be expressed in this system. Western blot and slot blot analysis revealed that although both the bifunctional enzyme protein and its mRNA were rapidly induced by the addition of isopropyl-1-thio-beta-D-galactopyranoside, the protein underwent rapid degradation. Deletion of the N-2 proline residue or its mutation to arginine, the corresponding residue in the rat liver enzyme, revealed that this proline residue was responsible for its rapid degradation. The Pro-2-->Arg mutant could be expressed with a high yield (20 mg/liter) in E. coli. The results support the hypothesis that a proline residue at N-2 facilitates bifunctional enzyme degradation in E. coli. The E. coli expressed mutant form was purified to homogeneity by phosphocellulose chromatography, and its kinetic properties were compared with those of the rat liver enzyme. The kinetic properties of the two enzymes were identical except for the presence of substrate (fructose 6-phosphate) inhibition of the human liver enzyme but not of the rat liver enzyme. The ability to express and purify large amounts of human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase will permit structure/function and x-ray crystal structure studies of the enzyme and ultimately its targeting for drug therapy.
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PMID:Expression of human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli. Role of N-2 proline in degradation of the protein. 838 36

We have previously constructed mutants of Saccharomyces cerevisiae in which the gene for the second-largest subunit of RNA polymerase I (Pol I) is deleted. In these mutants, rRNA is synthesized by RNA polymerase II from a hybrid gene consisting of the 35S rRNA coding region fused to the GAL7 promoter on a plasmid. These strains thus grow in galactose but not glucose media. By immunofluorescence microscopy using antibodies against the known nucleolar proteins SSB1 and fibrillarin, we found that the intact crescent-shaped nucleolar structure is absent in these mutants; instead, several granules (called mininucleolar bodies [MNBs]) that stained with these antibodies were seen in the nucleus. Conversion of the intact nucleolar structure to MNBs was also observed in Pol I temperature-sensitive mutants at nonpermissive temperatures. These MNBs may structurally resemble prenucleolar bodies observed in higher eukaryotic cells and may represent a constituent of the normal nucleolus. Furthermore, cells under certain conditions that inhibit rRNA synthesis did not cause conversion of the nucleolus to MNBs. Thus, the role of Pol I in the maintenance of the intact nucleolar structure might include a role as a structural element in addition to (or instead of) a functional role to produce rRNA transcripts. Our study also shows that the intact nucleolar structure is not absolutely required for rRNA processing, ribosome assembly, or cell growth and that MNBs are possibly functional in rRNA processing in the Pol I deletion mutants.
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PMID:Structural alterations of the nucleolus in mutants of Saccharomyces cerevisiae defective in RNA polymerase I. 845 21

Regulated transcription initiation requires, in addition to RNA polymerase II and the general transcription factors, accessory factors termed mediators or adapters. We have used affinity chromatography to identify a collection of factors that associate with Saccharomyces cerevisiae RNA polymerase II (P. A. Wade, W. Werel, R. C. Fentzke, N. E. Thompson, J. F. Leykam, R. R. Burgess, J. A. Jaehning, and Z. F. Burton, submitted for publication). Here we report identification and characterization of a gene encoding one of these factors, PAF1 (for RNA polymerase-associated factor 1). PAF1 encodes a novel, highly charged protein of 445 amino acids. Disruption of PAF1 in S. cerevisiae leads to pleiotropic phenotypic traits, including slow growth, temperature sensitivity, and abnormal cell morphology. Consistent with a possible role in transcription, Paf1p is localized to the nucleus. By comparing the abundances of many yeast transcripts in isogenic wild-type and paf1 mutant strains, we have identified genes whose expression is affected by PAF1. In particular, disruption of PAF1 decreases the induction of the galactose-regulated genes three- to fivefold. In contrast, the transcript level of MAK16, an essential gene involved in cell cycle regulation, is greatly increased in the paf1 mutant strain. Paf1p may therefore be required for both positive and negative regulation of subsets of yeast genes. Like Paf1p, the GAL11 gene product is found associated with RNA polymerase II and is required for regulated expression of many yeast genes including those controlled by galactose. We have found that a gal11 paf1 double mutant has a much more severe growth defect than either of the single mutants, indicating that these two proteins may function in parallel pathways to communicate signals from regulatory factors to RNA polymerase II.
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PMID:Paf1p, an RNA polymerase II-associated factor in Saccharomyces cerevisiae, may have both positive and negative roles in transcription. 855 95

Previous studies on a chromatin reporter gene (GAL-URARIB) in yeast showed that nucleosomes were maintained but rearranged during transcription in galactose, which was consistent with local dissociation of histones at the site of the RNA polymerase. Furthermore, repositioning of nucleosomes occurred rapidly after glucose repression. Because nucleosomal disruption and transcription produce topological changes in the chromatin substrate, the effect of topoisomerase activity was tested by the insertion of GAL-URABIB in topoisomerase mutant strains. The chromatin structure was analysed by nuclease digestion and psoralen crosslinking, and compared with that of the rDNA locus. In GAL-URARIB, neither the inactivation of topoisomerases I, II or I and II generated nucleosomal loss during transcription, nor was topoisomerase activity required for repositioning of the nucleosomes after repression. In contrast, the inactivation of topoisomerase I promoted an enhanced psoralen accessibility of the transcribed rDNA, possibly because of altered supercoiling, and the inactivation of topoisomerases I and II disrupted the chromatin structure of the whole rDNA locus by redistribution of the nucleosomes. The inactivation of topoisomerase II alone had no effect. These observations substantiate a differential participation of topoisomerases in the modulation of the chromatin structures of rDNA genes and of a single copy polymerase II gene. It is suggested that topological stress in genes transcribed by RNA polymerase II might diffuse away into flanking regions.
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PMID:Inactivation of topoisomerases affects transcription-dependent chromatin transitions in rDNA but not in a gene transcribed by RNA polymerase II. 859 42

CTLA4Ig, a fusion protein that blocks CD28-B7 costimulation, was studied in a LEW to F344 rat model of chronic cardiac rejection. In rats treated with a single dose of CTLA4Ig (0.5 mg intraperitoneally) 2 d after transplantation, allografts survived significantly longer ( > 70 d in 64%) than in untreated controls or rats treated with control Ig (all rejected within 25 d). Only 25% of grafts from rats treated with a single, high dose of cyclosporine A (25 mg/kg, 2 d after transplantation) survived longer than 70 d. Reverse transcriptase PCR and immunostaining analyses of tissue from 75-d, CTLA4Ig-treated allografts showed reduced expression of the T cell factor IFN-gamma and macrophage activation factors monocyte chemoattractant protein-1, inducible nitric oxide synthase, and galactose/N-acetylgalactosamine macrophage lectin, as well as TGF-beta. Grafts from longterm survivors ( > 120 d) treated with CTLA4Ig showed significant reductions in the frequency and severity of arteriosclerosis in comparison with cyclosporine A-treated rats. Thus, T cell activation is a proximal event in the cascade that culminates in the arteriosclerosis of chronic rejection. Strategies for blocking T cell costimulation may help prevent chronic rejection in clinical transplantation.
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PMID:Chronic cardiac rejection in the LEW to F344 rat model. Blockade of CD28-B7 costimulation by CTLA4Ig modulates T cell and macrophage activation and attenuates arteriosclerosis. 860 41

During transcription initiation from galP2, one of the two promoters of the Escherichia coli galactose operon with an initially transcribed sequence of pppAUUUC, RNA polymerase (RNAP) is known to engage nonproductive stuttering synthesis, which is sensitive to the concentration of UTP. This study examines the effect of this nonproductive synthesis on promoter clearance and determines other parameters that might affect stuttering synthesis by analyzing a mutant RNAP, RpoB3449, that has altered its function at this process at galP2. RpoB3449 has dramatically diminished stuttering synthesis, and consequently, it has increased the rate of productive initiation due to its enhanced rate of promoter clearance of galP2 compared with wild-type RNAP. Thus, a direct linkage between promoter clearance and productive transcription is demonstrated. The mechanism by which the mutant RNAP has altered the switch between nonproductive stuttering synthesis and productive initiation during promoter clearance is studied. Apparently, RpoB3449 has increased its efficiency in incorporating CTP at the +5 position of the galP2 transcript leading to its reduced stuttering synthesis, indicating that the rate of an RNAP incorporating the CTP after a stretch of uridine residues is important for promoter clearance at galP2. Because RpoB3449 demonstrates "wild-type" stuttering synthesis at the mutant galP2 promoter, which contains the 6 residue at the +5 position, it indicates that the mutant RNAP has altered in binding CTP at this context. Further experiments indicate that it is the +5 position per se of the galP2 sequence rather than a particular nucleotide at that position that is critical in determining the switch between the two alternate pathways during transcription initiation. A checkpoint model for the switch between nonproductive and productive initiations during promoter clearance is discussed.
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PMID:A mutant RNA polymerase reveals a kinetic mechanisms for the switch between nonproductive stuttering synthesis and productive initiation during promoter clearance. 866 41

Post-mitotic cultures of human mesangial cells were maintained in media containing 4-30 mM D-glucose for up to 28 days. Changes in mRNA and protein levels for specific macromolecules occurred between 7 and 14 days after initiating hyperglycaemic conditions. Slot blot analysis showed 2-3-fold increases in mRNAs for collagen type I, fibronectin, versican and perlecan, whereas mRNA for decorin was increased by up to 20-fold. Levels of mRNAs for biglycan and syndecan were unaffected by hyperglycaemic culture. Reverse transcriptase PCR (RT-PCR) confirmed that decorin mRNA levels are greatly elevated and also showed increased transcription of the TGF-beta 1 gene in hyperglycaemic cultures. Western analysis and ELISA indicated accumulations of collagen types I and III, laminin and fibronectin in the cell layers and media of hyperglycaemic cultures with increasing time. Type IV collagen did not accumulate in either compartment of hyperglycaemic mesangial cell cultures. Collagen types I, III, and fibronectin did not accumulate in the cell layers of hyperglycaemic human dermal fibroblasts, indicating a cell-specific response in mesangial cultures. Decorin and versican, but not biglycan, were increased in the hyperglycaemic mesangial cell culture media. There were no apparent changes in core proteins for decorin and biglycan in fibroblast media. Transforming growth factor beta 1 (TGF-beta 1) in hyperglycaemic mesangial cell cultures increased 5-fold after 7 days, but decreased thereafter to only approx. 2-fold after 28 days. The changes in TGF-beta 1 mRNA, as detected by RT-PCR, and protein followed one another closely.
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PMID:Expression of extracellular matrix molecules in human mesangial cells in response to prolonged hyperglycaemia. 867 Jan 79

Hydroxyl radical footprinting has been used to study different open complexes between Escherichia coli RNA polymerase and the galactose operon regulatory region, which contains two overlapping promoters, P1 and P2. Complexes at P1 were studied by exploiting a P2- mutant and complexes at P2 were studied with a P1-mutant. We have identified the precise location of alpha binding in both binary RNA polymerase-galP1 and RNA polymerase-P2 complexes from the effects of deletion of the C-terminal domain of the RNA polymerase alpha subunit: alpha binds to different sites at the upstream end of each complex. Transcription initiation at galP1 can be activated by the cyclic AMP receptor protein (CRP). Addition of CRP to the RNA polymerase-galP1 complex displaces the C-terminal domain of alpha, which then binds to a different site upstream of CRP in the ternary CRP-RNA polymerase-galP1 complex. Thus, the C-terminal domain of alpha can occupy three different sites at the gal operon regulatory region. We have also examined the effect of disrupting the Activating Region of CRP on interactions between CRP and the C-terminal domain of alpha in ternary CRP-RNA polymerase-galP1 complexes. Footprinting experiments show that these substitutions interfere with the contact between CRP and alpha but do not affect the position of alpha binding to its site upstream of bound CRP.
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PMID:Location of the C-terminal domain of the RNA polymerase alpha subunit in different open complexes at the Escherichia coli galactose operon regulatory region. 871 Apr 92

Gal4p regulates expression of genes necessary for galactose catabolism in Saccharomyces cerevisiae. We have previously shown that phosphorylation of Gal4p requires both its DNA binding and transcriptional-activation functions and have suggested that phosphorylation occurs as a consequence of interaction with general transcription factors. In this study, we show that phosphorylation occurs rapidly on a limited fraction of overexpressed Gal4p present in a sodium dodecyl sulfate-extractable subcellular fraction while a significant fraction remains stably unphosphorylated. Taken together with our previous observations, we conclude that Gal4p is phosphorylated only if it becomes localized to the nucleus and is capable of both DNA binding and transcriptional activation. We demonstrate that Gal4p is multiply phosphorylated at both the C and N termini, and we identify the precise locations of three sites of phosphorylation at serines 691, 696, and 699. Of these sites, only serine 699 must be phosphorylated for galactose-inducible transcription to occur. Mutation of S-699 to alanine significantly impairs GAL induction by galactose in GAL80+ cells but does not affect transcriptional activation by Gal4p in gal80- cells. In gal80- cells, Gal4p phosphorylation, including that of serine 699, is stimulated by the presence of both galactose and glucose, indicating that phosphorylation at this site is not specifically activated by galactose. Serine 699 phosphorylation requires Gal4p's DNA binding function and is influenced by the function of the RNA polymerase II holoenzyme component Gal11p. These results suggest that a phosphorylation on Gal4p, likely resulting from interaction with the holoenzyme, modulates the induction process by regulating interaction between Gal4p and Gal80p.
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PMID:Phosphorylation of Ga14p at a single C-terminal residue is necessary for galactose-inducible transcription. 875 47

Homologous recombination in Saccharomyces cerevisiae and other organisms can be stimulated by transcription. Consistent with this, we find that recombination of a chromosomal ade1 allele with a plasmid-borne ADE1 ORF under the control of the GAL1 promoter increased from 6.1x10(-6) to 1.7x10(-4) when transcription of the plasmid locus was induced by growing the cells in the presence of galactose. Recombination could also be stimulated by over-expressing the Gal4 transcription factor in the presence of the GAL1-ADE1 plasmid, while culturing the cells in dextrose medium. However, when transcription of the same ORF was driven from the highly active promoters of the rDNA (RNA polymerase I), and ADH1 (RNA polymerase II) genes, only background levels of recombination (5-10x10(-6)) were observed, irrespective of the carbon source. Recombination was found to involve integration of the whole plasmid and to depend on RAD51, RAD52 and RAD54. The results indicate that increased accessibility of transcriptionally active chromatin is not sufficient to cause increased rates of this kind of reciprocal exchange.
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PMID:Stimulation of mitotic recombination upon transcription from the yeast GAL1 promoter but not from other RNA polymerase I, II and III promoters. 892 89


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