Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P51532 (transcriptional activator)
 6,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the activation of eukaryotic heat shock genes, the acquisition of a binding ability to specific DNA sequence by a transcriptional activator, heat shock factor (HSF), is believed to be a crucial step. The induction of this new DNA binding activity of HSF is also obtained in a cell-free system (in vitro activation) by hyperthermia or at physiological temperature by calcium ions, low pH, urea, or non-ionic detergent. We report here the in vitro activation of HSF by treating at 0 degrees C a HeLa cell-free system with the aldehyde 4-hydroxynonenal (HNE), a highly cytotoxic product of lipid peroxidation. The in vitro activation of HSF by HNE occurred only if some components of the cell-free system were not sedimented at 100,000 x g. The reason for this is unclear but the release of active HSF from nuclei of unshocked cells and the involvement of Ca2+ contained in the mitochondria and ER have been excluded. Although HNE is known to be a sulfhydryl blocking agent, the results obtained with N-ethylmaleimide suggest that different mechanisms might be involved in the in vitro activation of HSF by HNE.
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PMID:In vitro activation of heat shock transcription factor by 4-hydroxynonenal. 139 24

RNA polymerase II lacking the fourth and seventh largest subunits (pol II delta 4/7) was purified from Saccharomyces cerevisiae strain rpb-4, in which the gene for the fourth largest subunit is deleted. pol II delta 4/7 was indistinguishable from wild-type pol II (holoenzyme) in promoter-independent initiation/chain elongation activity (400-800 nmol of nucleotide incorporated/10 min/mg of protein at 22 degrees C), in rate of chain elongation (20-25 nucleotides/s), and in the recognition of pause sites in the DNA template. In contrast to pol II holoenzyme, pol II delta 4/7 was inactive in promoter-directed initiation of transcription in vitro. The addition of an equimolar complex of the fourth and seventh largest subunits, purified from pol II holoenzyme by ion-exchange chromatography in the presence of urea, restored promoter-directed initiation activity to pol II delta 4/7. The transcriptional activator protein Gal4-VP16 could also elicit promoter-directed initiation by pol II delta 4/7 from a promoter with a Gal4 binding site. Complementation was observed between extracts of strain rpb-4, lacking the fourth largest subunit, and strain Y260-1, with a defect in the largest subunit. These extracts were individually inactive, but a mixture would support promoter-directed initiation. The fourth and seventh largest subunits may, therefore, shuttle between polymerase molecules.
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PMID:Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro. 198 24

The yeast Saccharomyces cerevisiae can use alternative nitrogen sources such as arginine, urea, allantoin, gamma-aminobutyrate, or proline when preferred nitrogen sources like glutamine, asparagine, or ammonium ions are unavailable in the environment. Utilization of alternative nitrogen sources requires the relief of nitrogen repression and induction of specific permeases and enzymes. The products of the GLN3 and URE2 genes are required for the appropriate transcription of many genes in alternative nitrogen assimilatory pathways. GLN3 appears to activate their transcription when good nitrogen sources are unavailable, and URE2 appears to repress their transcription when alternative nitrogen sources are not needed. The participation of nitrogen repression and the regulators GLN3 and URE2 in the proline utilization pathway was evaluated in this study. Comparison of PUT gene expression in cells grown in repressing or derepressing nitrogen sources, in the absence of the inducer proline, indicated that both PUT1 and PUT2 are regulated by nitrogen repression, although the effect on PUT2 is comparatively small. Recessive mutations in URE2 elevated expression of the PUT1 and PUT2 genes 5- to 10-fold when cells were grown on a nitrogen-repressing medium. Although PUT3, the proline utilization pathway transcriptional activator, is absolutely required for growth on proline as the sole nitrogen source, a put3 ure2 strain had somewhat elevated PUT gene expression, suggesting an effect of the ure2 mutation in the absence of the PUT3 product. PUT1 and PUT2 gene expression did not require the GLN3 activator protein for expression under either repressing or derepressing conditions. Therefore, regulation of the PUT genes by URE2 does not require a functional GLN3 protein. The effect of the ure2 mutation on the PUT genes is not due to increased internal proline levels. URE2 repression appears to be limited to nitrogen assimilatory systems and does not affect genes involved in carbon, inositol, or phosphate metabolism or in mating-type control and sporulation.
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PMID:Roles of URE2 and GLN3 in the proline utilization pathway in Saccharomyces cerevisiae. 789 26

A previous study (Govezensky, D., Greener, T., and Zamir, A. (1991) J. Bacteriol. 20, 6339-6346) indicated that the chaperonin GroEL was required for maximal expression from nif promoters in Klebsiella pneumoniae and nif-transformed Escherichia coli. That this requirement stemmed from the ability of GroEL to properly fold NifA, the nif transcriptional activator, was first supported by co-immunoprecipitation of NifA in K. pneumoniae extracts with anti-GroEL antibodies. In the present in vitro study, NifA, partially purified from E. coli overexpressing the protein, was diluted from a 6 M urea solution into a refolding buffer in the presence or absence of GroEL. Dilution in the absence of GroEL caused the complete precipitation of NifA. When present in the dilution buffer, GroEL bound NifA and maintained it in a soluble state. GroEL was also found to bind NifA newly synthesized in an in vitro translation system. For both NifA preparations, cochaperonin GroES and ATP promoted release of NifA from GroEL. These results provide evidence for the association of NifA with GroEL and for the role of both GroEL and GroES in the solubilization and thereby folding of the nif transcriptional activator.
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PMID:Chaperonins as potential gene regulatory factors. In vitro interaction and solubilization of NifA, the nif transcriptional activator, with GroEL. 791 Jun 8

Hepatocyte nuclear factor 4 (HNF-4), found in liver, kidney, and intestine, is a potent transcriptional activator that controls the expression of a wide variety of genes, including those involved in fatty acid and cholesterol metabolism, glucose metabolism, urea biosynthesis, blood coagulation, hepatitis B infections, and liver differentiation. HNF-4 is also a member of the steroid hormone receptor superfamily and has been highly conserved throughout evolution, suggesting that it might respond to an as yet unidentified ligand. In this presentation, some of the current findings regarding the role of HNF-4 in liver-specific gene expression are reviewed.
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PMID:Orphan receptor HNF-4 and liver-specific gene expression. 803 8

In the free-living diazotroph Klebsiella pneumoniae, the NifA protein is required for transcription of all nif (nitrogen fixation) operons except the regulatory nifLA operon itself. NifA activates transcription of nif operons by the alternative holoenzyme form of RNA polymerase, sigma 54 holoenzyme. In vivo, NifL is known to antagonize the action of NifA in the presence of molecular oxygen or combined nitrogen. We now demonstrate inhibition by NifL in vitro in both a coupled transcription-translation system and a purified transcription system. Crude cell extracts containing NifL inhibit NifA activity in the coupled system, as does NifL that has been solubilized with urea and allowed to refold. Inhibition is specific to NifA in that it does not affect activation by NtrC, a transcriptional activator homologous to NifA, or transcription by sigma 70 holoenzyme. Renatured NifL also inhibits transcriptional activation by a maltose-binding protein fusion to NifA in a purified transcription system, indicating that no protein factor other than NifL is required. Since inhibition in the purified system persists anaerobically, our NifL preparation does not sense molecular oxygen directly.
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PMID:In vitro activity of NifL, a signal transduction protein for biological nitrogen fixation. 824 38

The Proteus mirabilis and plasmid-encoded urease loci contain seven contiguous structural and accessory genes (ureDABCEFG) and the divergently transcribed ureR, which codes for an AraC-like transcriptional activator. Previously, it was shown that the plasmid-encoded ureR to ureD intergenic region contained divergent promoters (ureRp and ureDp). Transcription from these promoters required both the effector molecule urea and the activator protein UreR. In this report, we demonstrate that the P. mirabilis urease gene cluster contains similar divergent urea- and UreR-dependent promoters. The ureR gene products from either urease locus were able to activate transcription at both the plasmid-encoded and P. mirabilis promoters. The minimal concentration of urea required to activate transcription at ureRp or ureDp from either gene cluster was approximately 4 mM. The transcriptional start sites for the plasmid-encoded and P. mirabilis divergent promoters were similar in an Escherichia coli DH5 alpha background, as determined by primer-extension analysis. However, in P. mirabilis HI4320, transcription of ureR initiated predominately at an alternative site. Physical mapping and inhibition studies were used to localize the UreR-binding sites within the plasmid-encoded ureRp and ureDp intergenic sequences to regions of 68 bp and 86 bp, respectively. Gel shift analysis demonstrated that UreR bound to a 135 bp fragment in the approximate centre of the plasmid-encoded ureR to ureD intergenic region. The results presented here suggest that the P. mirabilis and plasmid-encoded urease gene clusters utilize similar mechanisms of transcriptional activation in response to urea.
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PMID:Activation of transcription at divergent urea-dependent promoters by the urease gene regulator UreR. 886 86

Proteus mirabilis, a cause of complicated urinary tract infection, expresses urease when exposed to urea. While it is recognized that the positive transcriptional activator UreR induces gene expression, the levels of expression of the enzyme during experimental infection are not known. To investigate in vivo expression of P. mirabilis urease, the gene encoding green fluorescent protein (GFP) was used to construct reporter fusions. Translational fusions of urease accessory gene ureD, which is preceded by a urea-inducible promoter, were made with gfp (modified to express S65T/V68L/S72A [B. P. Cormack et al. Gene 173:33-38, 1996]). Constructs were confirmed by sequencing of the fusion junctions. UreD-GFP fusion protein was induced by urea in both Escherichia coli DH5alpha and P. mirabilis HI4320. By using Western blotting with antiserum raised against GFP, expression level was shown to correlate with urea concentration (tested from 0 to 500 mM), with highest induction at 200 to 500 mM urea. Fluorescent E. coli and P. mirabilis bacteria were observed by fluorescence microscopy following urea induction, and the fluorescence intensity of GFP in cell lysates was measured by spectrophotofluorimetry. P. mirabilis HI4320 carrying the UreD-GFP fusion plasmid was transurethrally inoculated into the bladders of CBA mice. One week postchallenge, fluorescent bacteria were detected in thin sections of both bladder and kidney samples; the fluorescence intensity of bacteria in bladder tissue was higher than that in the kidney. Kidneys were primarily infected with single-cell-form fluorescent bacteria, while aggregated bacterial clusters were observed in the bladder. Elongated swarmer cells were only rarely observed. These observations demonstrate that urease is expressed in vivo and that using GFP as a reporter protein is a viable approach to investigate in vivo expression of P. mirabilis virulence genes in experimental urinary tract infection.
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PMID:Use of green fluorescent protein to assess urease gene expression by uropathogenic Proteus mirabilis during experimental ascending urinary tract infection. 942 75

The multifunctional 39 kDa Escherichia coli Ada protein (O6-methylguanine-DNA methyltransferase) (EC 2.1.1.63), product of the ada gene, is a monomeric globular polypeptide with two distinct alkylacceptor activities located in two domains. The two domains are of nearly equal size and are connected by a hinge region. The Ada protein accepts stoichiometrically the alkyl group from O6-alkylguanine in DNA at the Cys-321 residue and from alkyl phosphotriester at the Cys-69 residue. This protein functions in DNA repair by direct dealkylation of mutagenic O6-alkylguanine. The protein methylated at Cys-69 becomes a transcriptional activator of the genes in the ada regulon, including its own. Each of the two domains functions independently as an alkyl acceptor. The purified homogeneous protein is unstable at 37 degrees C and spontaneously loses about 30% of its secondary structure in less than 30 min concomitant with a complete loss of activity. However, sedimentation equilibrium studies indicated that the inactive protein remains in the monomeric form without aggregation. Furthermore, electrospray mass spectroscopic analysis indicated the absence of oxidation of the inactive protein. This temperature-dependent inactivation of the Ada protein is inhibited by DNA. In the presence of increasing concentrations of urea or guanidine, the protein gradually loses more than 80% of its structure. The two alkyl acceptor activities appear to be differentially sensitive to unfolding and the phosphotriester methyltransferase activity is resistant to 7 M urea. The partial or complete unfolding induced by urea or guanidine is completely reversed within seconds by removal of the denaturant. The heat-coagulated protein can also be restored to full activity by cycling it through treatment with 8 M urea or 6 M guanidine. These results suggest that the nascent or unfolded Ada polypeptide folds to a metastable form which is active and that the thermodynamically stable structure is partially unfolded and inactive.
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PMID:Reversible folding of Ada protein (O6-methylguanine-DNA methyltransferase) of Escherichia coli. 948 44

The closely related Proteus mirabilis and Enterobacterlaceae plasmid-encoded urease genes are positively regulated by the AraC-like transcriptional activator UreR. In the presence of the effector molecule urea, UreR promotes transcription of ureD, the initial gene in the urease operon, and increases transcription of the divergently transcribed ureR. Here, we identify UreR-specific binding sites in the ureRp-ureDp intergenic regions. Recombinant UreR (rUreR) was expressed and purified, and gel shift and DNase I protection assays were performed with this protein. These analyses indicated that there are two distinct rUreR binding sites in both the plasmid-encoded and P. mirabilis ureRp-ureDp intergenic regions. A consensus binding site of TA/GT/CA/TT/GC/TTA/TT/AATTG was predicted from the DNase I protection assays. Although rUreR bound to the specific DNA binding site in both the presence and the absence of urea, the dissociation rate constant k-1 of the rUreR-DNA complex interaction was measurably different when urea was present. In the absence of urea, the dissociation of the protein-DNA complexes, for both ureRp and ureDp, was complete at the earliest time point, and it was not possible to determine a rate. In the presence of urea, dissociation was measurable with a k-1 for the rUreR-ureRp interaction of 1.2 +/- 0.2 x 10(-2) s-1 and a k-1 for the rUreR-ureDp interaction of 2.6 +/- 0.1 x 10(-3) s-1. This corresponds to a half-life of the ureRp-rUreR interaction of 58 s, and a half-life of the ureDp-rUreR interaction of 4 min 26 s. A model describing a potential role for urea in the activation of these promoters is proposed.
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PMID:Identification of UreR binding sites in the Enterobacteriaceae plasmid-encoded and Proteus mirabilis urease gene operons. 1020 Sep 62


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