Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nitrogen regulatory protein C (NtrC) contacts a bacterial RNA polymerase from distant enhancers by means of DNA loops and activates transcription by allowing polymerase to gain access to the template DNA strand. It was shown that NtrC from Salmonella typhimurium must build large oligomers to activate transcription. In contrast to eukaryotic enhancer-binding proteins, most of which must bind directly to DNA, some NtrC dimers were bound solely by protein-protein interactions. NtrC oligomers were visualized with scanning force microscopy. Evidence of their functional importance was provided by showing that some inactive non-DNA-binding and DNA-binding mutant forms of NtrC can cooperate to activate transcription.
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PMID:Unusual oligomerization required for activity of NtrC, a bacterial enhancer-binding protein. 907 26

Activators of the sigma54-holoenzyme catalyze the isomerization of closed complexes between this polymerase and a promotor to open complexes in a reaction that depends upon hydrolysis of a nucleoside triphosphate. The activators normally bind to DNA sites with the properties of transcriptional enhancers and contact the polymerase by means of DNA loop formation. Here, we demonstrate that mutant forms of the activator nitrogen regulatory protein C (NtrC) that lack one helix of the helix-turn-helix (HTH) DNA-binding motif or the entire motif retain residual capacity to activate transcription from solution, despite the fact that they are largely unable to dimerize and have greatly decreased ability to hydrolyze ATP. We show that substitution of alanine for three hydrophilic residues in the second helix of the HTH yields a stable, dimeric form of NtrC defective in DNA-binding. Like mutant forms with deletions of one or both helices, the NtrC3ala protein failed to bind DNA in a sensitive affinity co-electrophoresis assay, indicating that its affinity for a strong enhancer was reduced by at least 5000-fold. (The assay detected enhancer-binding by two mutant forms of NtrC with single amino acid substitutions in the HTH and non-specific DNA-binding by the wild-type protein.) The phosphorylated NtrC3ala protein had normal ATPase activity in solution but, unlike the activity of the phosphorylated wild-type protein, which could be stimulated at least tenfold by an oligonucleotide carrying a strong enhancer, the ATPase activity of the phosphorylated NtrC3ala protein was not stimulated. At concentrations of 100 nM or greater, the phosphorylated NtrC3ala protein activated transcription from the major glnA promoter. In agreement with the fact that it did not show detectable DNA-binding in other assays, its ability to activate transcription was no greater on templates carrying the glnA enhancer than on templates lacking an enhancer. The results indicate that both roles of the glnA enhancer, tethering and facilitation of the formation of an active oligomer of NtrC, can be bypassed if the protein is present at high concentrations in solution.
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PMID:Mutant forms of the enhancer-binding protein NtrC can activate transcription from solution. 909 4

Scanning force microscopy (SFM) has been used to study transcriptional activation of Escherichia coli RNA polymerase x sigma 54 (RNAP x sigma 54) at the glnA promoter by the constitutive mutant NtrC(D54E,S160F) of the NtrC Protein (nitrogen regulatory protein C). DNA-protein complexes were deposited on mica and images were recorded in air. The DNA template was a 726 bp linear fragment with two NtrC binding sites located at the end and about 460 bp away from the RNAP x sigma 54 glnA promoter. By choosing appropriate conditions the structure of various intermediates in the transcription process could be visualized and analyzed: (1) different multimeric complexes of NtrC(D54E,S160F) dimers bound to the DNA template; (2) the closed complex of RNAP x sigma 54 at the glnA promoter; (3) association between DNA bound RNAP x sigma 54 and NtrC(D54E,S160F) with the intervening DNA looped out; and (4) the activated open promoter complex of RNAP x sigma 54. Measurements of the DNA bending angle of RNAP x sigma 54 closed promoter complexes yielded an apparent bending angle of 49(+/-24) degrees. Under conditions that allowed the formation of the open promoter complex, the distribution of bending angles displayed two peaks at 50(+/-24) degrees and 114(+/-18) degrees, suggesting that the transition from the RNAP x sigma 54 closed complex to the open complex is accompanied by an increase of the DNA bending angle.
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PMID:Transcriptional activation via DNA-looping: visualization of intermediates in the activation pathway of E. coli RNA polymerase x sigma 54 holoenzyme by scanning force microscopy. 923 16

The transcription activator protein NtrC (nitrogen regulatory protein C) can catalyze the transition of E. coli RNA polymerase complexed with the sigma54 factor (RNAP.sigma54) from the closed complex (RNAP.sigma54 bound at the promoter) to the open complex (melting of the promoter DNA). This process involves phosphorylation of NtrC, assembly of a multimeric NtrC complex at the enhancer DNA sequence, interaction of this complex with promoter bound RNAP. sigma54 via DNA looping, and hydrolysis of ATP. We have used analytical ultracentrifugation to study the different NtrC association states and to derive hydrodynamic models for the conformation of the various NtrC species. The following results were obtained. (i) The unphosphorylated wild-type protein formed a dimer with a measured molecular weight of 102(+/-3) kDa, which compares to a calculated molecular weight of 54 kDa for a monomer (concentration range studied 2 to 8 microM NtrC monomer). (ii) In the unphosphorylated state one NtrC dimer was bound to one binding site as determined with DNA oligonucleotide duplexes containing one or two binding sites (concentration range studied 50 to 1000 nM NtrC dimer). (iii) The data obtained at protein concentrations that were below the concentration of binding sites indicate that binding to the DNA duplex with two binding sites occurred with essentially no cooperativity. The experiments were conducted in the absence of ATP. (iv) The phosphorylated protein formed a specific complex at the DNA duplex with the enhancer sequence (two NtrC binding sites) that consisted of four dimers (concentration range studied 100 to 1000 nM NtrC dimer). (v) The formation of this octameric complex was highly cooperative, and the data suggest that two DNA strands could bind simultaneously to this complex. (vi) From the sedimentation data a model was derived in which the NtrC dimer adopts a V shaped structure with the DNA binding domains being located at the bottom and the two receiver domains at the top of the V. In this conformation higher order NtrC complexes can be stabilized by interaction between the phosphorylated receiver domain and the central activation domain of different NtrC dimers.
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PMID:Association states of the transcription activator protein NtrC from E. coli determined by analytical ultracentrifugation. 960 Aug 53

There are many reports supporting a self-limitation mechanism involved with hypermetabolic response after severe cranial injury. It was proposed a study with severe head injury patients, in three stages of the evolution. The first 7 days after admission (moment 1-M1), the second three days latter (M2) and the last 7 days after the first (M3). Among male patients with severe head injury, attended between January 1992 and December 1993 in University Hospital of Botucatu, UNESP, were selected 28 male patients, with Glasgow severity scale between 4 and 6, with pO2 < 70 mm Hg, weighting 60 kg or more. Among these patients, 6 finished the study, including analysis of the excretion of N, acute phase proteins, glycemia, triglycerides and amine nitrogen. During the study there were no changes in nitrogen balance and there was a decrease in protein C-reative. Glycemia tends to fall within two weeks after injury. The authors make some considerations about possible mechanisms involved in brain modulation associated with the period of dependence of hypermetabolism and hypercatabolism after closed brain injury. There are some evidences that the brain responds to head trauma with a gobal non specific way, which tends to be reorganized beyond the first two weeks after lesion. The study does not show any influence of the type and severity of head trauma.
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PMID:[Is the metabolic response self-limited in head trauma? Analysis of acute phase proteins and glycemia]. 1002 82

The bacterial enhancer-binding protein nitrogen regulatory protein C (NtrC) activates transcription by sigma54-containing RNA polymerase in a reaction that depends on ATP hydrolysis. Phosphorylation of an aspartate residue in the N-terminal receiver domain of NtrC induces oligomerization of the protein and activates the ATPase activity, which is a function of its central output domain. To study the role of the receiver domain of NtrC, which is known to act positively, we isolated mutant forms of the protein carrying single cysteine residues and derivatized them with a sulfhydryl-specific nitroxide reagent for electron paramagnetic resonance studies. Single cysteines were placed at four positions at which we had obtained constitutive amino acid substitutions, those that yield activity without phosphorylation. In only one case, derivatized C86 in alpha-helix 4 of the receiver domain, did the motion of the side chain become dramatically slower upon phosphorylation. Importantly, derivatized NtrCD86C (NtrCD86C*) activated transcription normally. Additional experiments indicated that the spectral change observed upon phosphorylation of NtrCD86C* was due to interdomain interactions rather than a conformational change within the N-terminal domain itself. These interactions did not appear to occur within a monomer. Although it is not clear whether the spectral change seen upon phosphorylation of NtrCD86C* is due to an interaction that occurs within a dimer of NtrC or requires the formation of higher-order oligomers, the change indicated that alpha-helix 4 of the receiver domain probably plays an important role in communication with the remainder of the protein.
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PMID:Physical evidence for a phosphorylation-dependent conformational change in the enhancer-binding protein NtrC. 1022 Mar 87

When phosphorylated, the dimeric form of nitrogen regulatory protein C (NtrC) of Salmonella typhimurium forms a larger oligomer(s) that can hydrolyze ATP and hence activate transcription by the sigma(54)-holoenzyme form of RNA polymerase. Studies of Mg-nucleoside triphosphate binding using a filter-binding assay indicated that phosphorylation is not required for nucleotide binding but probably controls nucleotide hydrolysis per se. Studies of binding by isothermal titration calorimetry indicated that the apparent K(d) of unphosphorylated NtrC for MgATPgammaS is 100 microM at 25 degrees C, and studies by filter binding indicated that the concentration of MgATP required for half-maximal binding is 130 microM at 37 degrees C. Filter-binding studies with mutant forms of NtrC defective in ATP hydrolysis implicated two regions of its central domain directly in nucleotide binding and three additional regions in hydrolysis. All five are highly conserved among activators of sigma(54)-holoenzyme. Regions implicated in binding are the Walker A motif and the region around residues G355 to R358, which may interact with the nucleotide base. Regions implicated in nucleotide hydrolysis are residues S207 and E208, which have been proposed to lie in a region analogous to the switch I effector region of p21(ras) and other purine nucleotide-binding proteins; residue R294, which may be a catalytic residue; and residue D239, which is the conserved aspartate in the putative Walker B motif. D239 appears to play a role in binding the divalent cation essential for nucleotide hydrolysis. Electron paramagnetic resonance analysis of Mn(2+) binding indicated that the central domain of NtrC does not bind divalent cation strongly in the absence of nucleotide.
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PMID:MgATP binding and hydrolysis determinants of NtrC, a bacterial enhancer-binding protein. 1041 63

NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein of 469 residues that activates transcription by sigma(54)-holoenzyme. A region of its transcriptional activation (central) domain that is highly conserved among homologous activators of sigma(54)-holoenzyme-residues 206-220-is essential for interaction with this RNA polymerase: it is required for contact with the polymerase and/or for coupling the energy from ATP hydrolysis to a change in the conformation of the polymerase that allows it to form transcriptionally productive open complexes. Several mutant NtrC proteins with amino acid substitutions in this region, including NtrC(A216V) and NtrC(G219K), have normal ATPase activity but fail in transcriptional activation. We now report that other mutant forms carrying amino acid substitutions at these same positions, NtrC(A216C) and NtrC(G219C), are capable of activating transcription when they are not bound to a DNA template (non-DNA-binding derivatives with an altered helix-turn-helix DNA-binding motif at the C terminus of the protein) but are unable to do so when they are bound to a DNA template, whether or not it carries a specific enhancer. Enhancer DNA remains a positive allosteric effector of ATP hydrolysis, as it is for wild-type NtrC but, surprisingly, appears to have become a negative allosteric effector for some aspect of interaction with sigma(54)-holoenzyme. The conserved region in which these amino acid substitutions occur (206-220) is equivalent to the Switch I region of a large group of purine nucleotide-binding proteins. Interesting analogies can be drawn between the Switch I region of NtrC and that of p21(ras).
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PMID:"Switch I" mutant forms of the bacterial enhancer-binding protein NtrC that perturb the response to DNA. 1055 87

Receiver domains are the dominant molecular switches in bacterial signalling. Although several structures of non-phosphorylated receiver domains have been reported, a detailed structural understanding of the activation arising from phosphorylation has been impeded by the very short half-lives of the aspartylphosphate linkages. Here we present the first structure of a receiver domain in its active state, the phosphorylated receiver domain of the bacterial enhancer-binding protein NtrC (nitrogen regulatory protein C). Nuclear magnetic resonance spectra were taken during steady-state autophosphorylation/dephosphorylation, and three-dimensional spectra from multiple samples were combined. Phosphorylation induces a large conformational change involving a displacement of beta-strands 4 and 5 and alpha-helices 3 and 4 away from the active site, a register shift and an axial rotation in helix 4. This creates an exposed hydrophobic surface that is likely to transmit the signal to the transcriptional activation domain.
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PMID:Structure of a transiently phosphorylated switch in bacterial signal transduction. 1062 55

The transcription activator protein NtrC (nitrogen regulatory protein C, also termed NR(I)) can catalyze the transition of Escherichia coli RNA polymerase complexed with the sigma(54) factor (RNAP x sigma(54)) from the closed complex (RNAP x sigma(54) bound at the promoter) to the open complex (melting of the promoter DNA). This process involves phosphorylation of NtrC (NtrC-P), assembly of an octameric NtrC-P complex at the enhancer DNA sequence, interaction of this complex with promoter-bound RNAP x sigma(54) via DNA looping, and hydrolysis of ATP. Here it is demonstrated by two-color fluorescence cross-correlation spectroscopy measurements of 6-carboxyfluorescein and 6-carboxy-X-rhodamine-labeled DNA oligonucleotide duplexes that the NtrC-P complex can bind two DNA duplexes simultaneously. This suggests a model for the conformation of the looped intermediate that is formed between NtrC-P and RNAP. sigma(54) at the glnAp2 promoter during the activation process.
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PMID:Simultaneous binding of two DNA duplexes to the NtrC-enhancer complex studied by two-color fluorescence cross-correlation spectroscopy. 1069 78


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