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

---

Query: EC:2.7.7.6 (RNA polymerase)
34,946 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.
...
PMID:Unusual oligomerization required for activity of NtrC, a bacterial enhancer-binding protein. 907 26

We had earlier overproduced the transcription activator protein C of bacteriophage Mu in a phage-T7 expression system. Although we achieved a high level of overproduction, the expression was not consistent. This could be due to the leaky expression of T7 RNA polymerase in the uninduced state. Introduction of pLysS, a plasmid encoding T7 lysozyme, a natural inhibitor of T7 RNA polymerase, resulted in consistent, but extremely low production of the C protein. To overcome this problem, we have devised an artificial regulatory circuit to obtain stabilised, consistent overproduction of C protein. The C-binding site was cloned downstream from the transcription start point of T7 lys. Upon induction, the C protein produced binds to its site with a very high affinity, possibly acting as a transcriptional roadblock for lys. This would overcome the inhibitory effect of T7 lysozyme on T7 RNA polymerase.
...
PMID:An artificial regulatory circuit for stable expression of DNA-binding proteins in a T7 expression system. 918 43

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.
...
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

IL-12, a 75-kDa heterodimeric cytokine composed of two chains (p35 and p40), is a central regulator of immune responses and may be implicated in the pathogenesis of certain inflammatory diseases of the central nervous system (CNS). We have examined the capacity of two CNS APC, microglia and astrocytes, to produce IL-12 upon stimulation with cytokines, LPS, or a neurotropic virus. In purified microglial cultures from neonatal mouse brains, expression of IL-12 p35 and p40 mRNA is induced by LPS and is stimulated maximally by combined IFN-gamma/LPS treatment, as detected by semiquantitative reverse-transcriptase PCR. LPS induces secretion of IL-12 p40, but not of IL-12 p75, as detected by specific ELISA. Combined stimulation with IFN-gamma/LPS enhances IL-12 p40 secretion and induces IL-12 p75 secretion by microglia. Conversely, mouse astrocytes do not express IL-12 p35 mRNA and do not secrete IL-12 p75 under any condition tested. IL-12 production by activated microglia is inhibited by IL-10, PGE2, and cAMP-elevating agents. Coculture of microglia with astrocytes or exposure of microglia to astrocyte-conditioned medium also results in marked reduction of IL-12 p75 and p40 secretion by IFN-gamma/LPS-stimulated microglia, indicating a regulatory role of astrocytes on IL-12 production. This novel mechanism of IL-12 regulation may play an important role in the control of immune responses during infection or in Th1 cell-mediated autoimmune diseases of the CNS.
...
PMID:IL-12 production by central nervous system microglia is inhibited by astrocytes. 925 19

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.
...
PMID:Association states of the transcription activator protein NtrC from E. coli determined by analytical ultracentrifugation. 960 Aug 53

We have developed a new strategy with a very tight control for the expression of cloned genes. The system employed here is the T7 promoter-based expression system in which transcription activator protein C of bacteriophage Mu (Mu C) has been cloned to serve as a repressor in the regulatory circuit. The system also includes pLysE, which encodes T7 lysozyme, an inhibitor of T7 RNA polymerase. This ensures tight regulation of cloned genes in the uninduced state. Upon induction, the expressed Mu C protein binds to its cognate site thereby repressing lys transcription driven by the tet promoter. In order to evaluate the tight control achieved in the system, and to check leaky expression, if any, we have cloned the gene for the SmaI restriction endonuclease without its cognate methylase. For this purpose, a dicistronic unit was constructed by cloning the smaIR gene downstream of the Mu C gene. SmaI expression was observed only in the induced cell extracts, demonstrating a tight control. The system could be used to express the genes of other cloned restriction enzymes and has the potential for general applications.
...
PMID:Design of a novel regulatory circuit for expression of restriction endonucleases. 962 59

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.
...
PMID:Physical evidence for a phosphorylation-dependent conformational change in the enhancer-binding protein NtrC. 1022 Mar 87

The nucleotide sequence of the entire gene encoding the murine endothelial cell receptor for activated protein C (EPCR) has been determined. A total of 5303 bp of DNA was sequenced that included 4 exons and three introns, which constituted the coding region of the gene, as well as 393 bp upstream of the first exon and 841 bp downstream of the last exon. From the locations of the introns in this gene and analysis of the exon structures, it is clear the EPCR gene is a member of the CD1 class of multiple histocompatibility proteins. and its cDNA sequence is nearly identical to that of CCD41, a centrosome-associated protein. All elements needed for RNA polymerase II-based transcription are predicted to exist in the 5' uncoded region of the gene, and potential 3' regulatory sequences for efficient polyadenylation have been located at their optimal locations. A variety of highly probable transcription factor binding sites have been located in the 5' region of the gene. These data suggest that the EPCR gene is under efficient transcriptional control, and support the finding that this gene product may be involved in the inflammatory pathway.
...
PMID:Nucleotide structure and characterization of the murine gene encoding the endothelial cell protein C receptor. 1023 44

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.
...
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).
...
PMID:"Switch I" mutant forms of the bacterial enhancer-binding protein NtrC that perturb the response to DNA. 1055 87


<< Previous 1 2 3 4 5 Next >>

---