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

Rhizobium meliloti DctD (C4-dicarboxylate transport protein D) is a transcriptional activator that catalyzes the ATP-dependent isomerization of closed complexes between sigma 54-RNA polymerase holoenzyme and the dctA promoter to open complexes. Following random mutagenesis of dctD, 55 independent mutant forms of DctD that failed to activate transcription from a dctA'-'lacZ reporter gene in Escherichia coli were selected, and the amino acid substitutions were determined for these mutant proteins. Amino acid substitutions were distributed throughout the central domain of the protein, the domain responsible for transcription activation, but most of the substitutions occurred within three highly conserved regions of the protein. Selected mutant proteins were purified, and their activities were studied in vitro. All of the purified mutant proteins appeared to have normal DNA-binding activity and interacted with sigma 54 and core RNA polymerase, as determined from protein crosslinking assays. Proteins with amino acid substitutions in a region spanning amino acid positions 222 to 225 retained their ATPase activities, whereas proteins with substitutions in other regions had little or no ATPase activity. Taken together, these data suggest that the region that encompasses amino acid residues 222 through 225 probably functions in coupling the energy released from ATP hydrolysis to open complex formation rather than as a major determinant for binding to RNA polymerase.
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PMID:Alterations within the activation domain of the sigma 54-dependent activator DctD that prevent transcriptional activation. 929 39

The INS-r3-GK27 insulinoma cells are endowed with artificially inducible glucokinase under control of the reverse tetracycline-dependent transcriptional activator. Moderate induction of glucokinase has been shown to result in proportionate increases in glycolytic flux and in potentiation of glucose effects on insulin secretion and pyruvate kinase gene expression. In cells with 20-fold overexpression of glucokinase, however, glucose activation of secretion and gene expression was severely impaired. Measurements of the glycolytic flux in cells with 7- and 21-fold increases in glucokinase activity and determination of the flux control coefficient of this enzyme showed that control of glycolysis at the glucokinase step was lost in the cells at the higher level of overexpression. Challenging the cells with glucose above 6 mM resulted in massive accumulation of glucose 6-phosphate and caused a rapid and sustained depletion of cellular ATP, in contrast with the glucose-induced rise in ATP in cells with wild-type glucokinase levels. Loss of cell viability ensued upon prolonged culture in high glucose. In summary, in insulinoma beta cells strongly overexpressing glucokinase, an imbalance between glucose phosphorylation and turnover of glucose 6-phosphate resulted in acute glucose intolerance due to trapping of cellular orthophosphate in dead-end product and severe paralysis of energy metabolism.
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PMID:Acute glucose intolerance in insulinoma cells with unbalanced overexpression of glucokinase. 932 99

By controlled addition of galactose to synchronized galactose-limited Saccharomyces cerevisiae cultures, the growth rate could be regulated while external conditions were kept constant. By using this method, the G1 phase duration was modulated and expression of cell cycle-regulated genes was investigated. The expression of the cyclin genes CLN1 and CLN2 was always induced just before bud emergence, indicating that this event marks the decision to pass Start. Thus, G1 phase elongation was not due to a slower accumulation of the CLN1 and CLN2 mRNA levels. Only small differences in CLN3 expression levels were observed. The maximal SWI4 expression preceded maximal CLN1 and CLN2 expression under all conditions, as expected for a transcriptional activator. But whereas SWI4 was expressed at about 10 to 20 min, before CLN1 and CLN2 expression at high growth rates, this time increased to about 300 min below a particular consumption rate at which the G1 phase strongly elongated. In the slower-growing cultures, also an increase in SWI6 expression was observed in the G1 phase. The increase in G1 phase duration below a particular consumption rate was accompanied by a strong increase in the reserve carbohydrate levels. These carbohydrates were metabolized again before bud emergence, indicating that below this consumption rate, a transient increase in ATP flux is required for progression through the cell cycle. Since Start occurred at different cell sizes under different growth conditions, it is not just a certain cell size that triggers passage through Start.
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PMID:Effects of different carbon fluxes on G1 phase duration, cyclin expression, and reserve carbohydrate metabolism in Saccharomyces cerevisiae. 935

Azotobacter vinelandii NIFL is a nitrogen fixation-specific regulatory flavoprotein that modulates the activity of the transcriptional activator NIFA in response to oxygen and fixed nitrogen in vivo. NIFL is also responsive to ADP in vitro. Limited proteolysis of NIFL indicates that it comprises a relatively stable N-terminal domain and a C-terminal domain that is protected from trypsin digestion in the presence of adenosine nucleotides. ATP protects the protein from cleavage in the vicinity of potential nucleotide-binding sites in the C-terminus, whereas ADP protects the entire C-terminal domain. NIFL has an apparent Kd of 130 microM for ATP and 16 microM for ADP. The purified N-terminal domain has an identical UV/visible absorption spectrum to the wild-type protein and is reduced by sodium dithionite, demonstrating that it is a flavin-binding domain. The isolated N-terminal domain does not inhibit NIFA activity. A subdomain fragment containing 160 residues of the C-terminal region, including the nucleotide-binding sites, is also not competent to inhibit NIFA. Removal of the first 146 residues of NIFL, which includes a conserved S-motif (PAS-like domain), found in a large family of sensory proteins from eubacteria, archea and eukarya eliminates the redox response. However, this truncated protein remains competent to inhibit NIFA activity in response to ADP in vitro and to the level of fixed nitrogen in vivo. The redox and nitrogen-sensing functions of A. vinelandii NIFL are therefore separable and are discrete functions of the protein.
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PMID:The redox- and fixed nitrogen-responsive regulatory protein NIFL from Azotobacter vinelandii comprises discrete flavin and nucleotide-binding domains. 959 6

In the purple non-sulfur bacterium Rhodobacter capsulatus, RegA and RegB comprise a two-component regulatory system that is required for maximal anaerobic transcription of key photosynthesis genes. RegB is a sensor kinase that uses ATP to phosphorylate its cognate response regulator, RegA. The mechanism under which RegA approximately P influences transcription of target genes has been unclear given that past attempts to demonstrate DNA binding activity by isolated RegA have failed. This led to a model invoking a role for RegA approximately P as an intermediate in a more complex multicomponent phosphoryl transfer cascade. In the present study, we describe the isolation of a mutant version of RegA (RegA*) which promotes high level expression of photosynthesis genes independent of RegB. DNase I footprint analyses show that purified RegA* binds to the promoters of the puf and puc operons at locations that are consistent with RegA functioning as a transcriptional activator for these operons. We conclude that RegA functions, like most members of the response regulator family, as a DNA-binding protein that directly affects the expression of its target genes.
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PMID:DNA binding characteristics of RegA. A constitutively active anaerobic activator of photosynthesis gene expression in Rhodobacter capsulatus. 966 Aug 20

MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.
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PMID:Degradation of myogenic transcription factor MyoD by the ubiquitin pathway in vivo and in vitro: regulation by specific DNA binding. 974 84

The ftsH gene encodes an ATP- and Zn(2+)-dependent metalloprotease with a molecular mass of about 70 kDa. It was first identified in Escherichia coli where it is also designated hflB, tolZ or mrsC, and seems to be present in most if not all bacteria. The FtsH protein is anchored to the cytoplasmic membrane via two transmembrane regions in such a way that the very short amino- and the long carboxy-termini are exposed into the cytoplasm. FtsH is member of the AAA family (ATPases associated with a variety of cellular activities) which are characterized by a module of about 200 amino acid residues in length containing an ATP-binding site. In Escherichia coli, FtsH forms a complex with a pair of periplasmically exposed membrane proteins, HflK and HflC. The E. coli enzyme is required for proteolytic degradation of some unstable proteins that include both soluble regulatory proteins such as sigma 32 (heat-shock sigma factor) and phage lambda CII (transcriptional activator), and membrane proteins including uncomplexed forms of SecY (forms the translocon together with SecE and SecG) and the a subunit of the F0 complex of the H(+)-ATPase. Its activity can be modulated by the HflKC proteins, by another membrane protein designated YccA which can transiently associate with both the FtsH and the HflKC proteins, or by small peptides such as CIII encoded by phage lambda (involved in lysogenization) or SpoVM (needed for sporulation) encoded by Bacillus subtilis. Besides being a protease, there is circumstantial evidence that FtsH also acts as a molecular chaperone. It influences protein assembly in and through the cytoplasmic membrane and associates with denatured alkaline phosphatase without degrading it. Therefore, FtsH may serve to maintain quality control of some cytoplasmic and membrane proteins. Such ATP-dependent proteases with intrinsic chaperone activity have been designated charonins.
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PMID:FtsH--a single-chain charonin? 1007 51

Pseudomonas aeruginosa, when deprived of oxygen, generates ATP from arginine catabolism by enzymes of the arginine deiminase pathway, encoded by the arcDABC operon. Under conditions of low oxygen tension, the transcriptional activator ANR binds to a site centered 41.5 bp upstream of the arcD transcriptional start. ANR-mediated anaerobic induction was enhanced two- to threefold by extracellular arginine. This arginine effect depended, in trans, on the transcriptional regulator ArgR and, in cis, on an ArgR binding site centered at -73.5 bp in the arcD promoter. Binding of purified ArgR protein to this site was demonstrated by electrophoretic mobility shift assays and DNase I footprinting. This ArgR recognition site contained a sequence, 5'-TGACGC-3', which deviated in only 1 base from the common sequence motif 5'-TGTCGC-3' found in other ArgR binding sites of P. aeruginosa. Furthermore, an alignment of all known ArgR binding sites confirmed that they consist of two directly repeated half-sites. In the absence of ANR, arginine did not induce the arc operon, suggesting that ArgR alone does not activate the arcD promoter. According to a model proposed, ArgR makes physical contact with ANR and thereby facilitates initiation of arc transcription.
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PMID:The ArgR regulatory protein, a helper to the anaerobic regulator ANR during transcriptional activation of the arcD promoter in Pseudomonas aeruginosa. 1019 9

MyoD is a tissue-specific transcriptional activator involvd in skeletal muscle differentiation. It is induced during transition from proliferating, non-differentiated myoblasts to the resting and well differentiated myotubes. Like many other transcriptional regulators, it is short-lived, however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved have remained obscure. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In cells, degradation is inhibited by lactacystin, a specific inhibitor of the 20S proteasome. Inhibition is accompanied by accumulation of MyoD-ubiquitin conjugates. In a cell free system, the proteolytic process requires both ATP and ubiquitin and is preceded by formation of MyoD-ubiquitin adducts. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds. Analysis of the ubiquitination site has revealed that the N-terminal residue of MyoD is sufficient and essential to promote conjugation and subsequent degradation of the protein: conjugation to internal Lys residues is not necessary. Substitution of all Lys residues did not affect significantly its degradation either in intact cells or in a reconstituted cell free system. Degradation was inhibited by specific proteasome inhibitors and was accompanied by accumulation of ubiquitinated species of the protein. We concluded that the first ubiquitin moiety is attached via its C-terminal Gly to the N-terminal residue of MyoD, and the polyubiquitin chain is then synthesized on Lys48 of this moiety.
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PMID:Degradation of MyoD by the ubiquitin pathway: regulation by specific DNA-binding and identification of a novel site for ubiquitination. 1036 48

Under low oxygen conditions, induction of many genes required for nitrogen fixation in Bradyrhizobium japonicum depends on the redox-responsive transcriptional activator NifA which is encoded in the fixR-nifA operon. Basal expression of this operon depends on the response regulator RegR and a DNA element located around position -68 in the fixR-nifA promoter region. To investigate the functional properties of RegR and the interaction with its putative cognate kinase, RegS, we overproduced and affinity-purified RegR and a truncated soluble variant of RegS (RegS(C)), both as N-terminally His(6)-tagged proteins. RegS(C) autophosphorylated when incubated with [gamma-(32)P]ATP, and it catalyzed the transfer of the phosphoryl label to RegR. The phosphorylated form of RegS(C) exhibited phosphatase activity on RegR-phosphate. Chemical stability tests and site-specific mutagenesis identified amino acids H219 and D63 of RegS and RegR, respectively, as the phosphorylated residues. Competition experiments with isolated domains demonstrated that the N-terminal but not the C-terminal domain of RegR interacts with RegS(C). Band-shift experiments revealed that phosphorylated RegR had at least eightfold enhanced DNA-binding activity compared with dephosphorylated RegR or the mutant protein RegR-D63N, which cannot be phosphorylated. In conclusion, the RegSR proteins of B. japonicum exhibit functional properties in vitro that are typical of two-component regulatory systems.
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PMID:Phosphorylation, dephosphorylation and DNA-binding of the Bradyrhizobium japonicum RegSR two-component regulatory proteins. 1040 54


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