Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q02556 (DNA-binding domain)
 6,431 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In bacteria and lower eukaryotes, adaptation to changes in the environment is often mediated by two-component regulatory systems. Such systems provide the basis for chemotaxis, nitrogen and phosphate regulation and adaptation to osmotic stress, for example. In Escherichia coli, the sensor kinase EnvZ detects a change in the osmotic environment and phosphorylates the response regulator OmpR. Phospho-OmpR binds to the regulatory regions of the porin genes ompF and ompC, and alters their expression. Recent evidence suggests that OmpR functions as a global regulator, regulating additional genes besides the porin genes. In this study, we have characterized a previously isolated OmpR2 mutant (V203M) that constitutively activates ompF and fails to express ompC. Because the substitution was located in the C-terminal DNA-binding domain, it had been assumed that the substitution would not affect phosphorylation of the N-terminal domain of OmpR. Our results indicate that this substitution completely eliminates phosphorylation by a small phosphate donor, acetyl phosphate, but not phosphorylation by the kinase EnvZ. The mutant OmpR has altered dephosphorylation kinetics and altered binding affinities to both ompF and ompC sites compared to the wild-type. Thus, a single amino acid substitution in the C-terminal DNA-binding domain has dramatic effects on the N-terminal phosphorylation domain. Most strikingly, we have identified a single base change in the OmpR binding site of ompC that restores high-affinity binding activity by the mutant. We interpret our results in the context of a model for porin gene expression.
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PMID:A single amino acid substitution in the C terminus of OmpR alters DNA recognition and phosphorylation. 1087 50

Regulation of aflatoxin (AF) biosynthesis likely involves a complex interplay of positive- and negative-acting factors that are affected by physiological cues responsive to internal and external stimuli. These factors, presumably, modulate the expression of the AF pathway-specific regulatory gene, aflR, whose product, AFLR, a zinc cluster transcription factor, then turns on or off the transcription of other AF genes. To determine if the AFLR carboxyl region (AFLRC) interacts with positive- or negative-acting proteins, we fused the Aspergillus parasiticus aflR carboxyl coding region (aflRC) to the promoter of A. parasiticus nitrite reductase gene (niiA(p)::aflRC), and transformed it into A. parasiticus SRRC 2043. Transformants that contained two copies of niiA(p)::aflRC, one at the niaD locus and another at the aflR locus, overproduced AF precursors independent of the nitrogen source. The higher copy number of the integrated niiA(p)::aflRC correlated with increased production of AF precursors by the transformants as well as increased expression of both aflRC and native aflR in potato dextrose broth and A&M medium. Since aflRC does not encode a DNA-binding domain, the expressed AFLRC should not bind to the promoters of AF pathway genes and affect transcription directly. The results are consistent with AFLRC titrating out a putative repressor that interacts with AFLR under different growth conditions and modulates AF biosynthesis. This interaction also indirectly affects sclerotial development.
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PMID:Repressor-AFLR interaction modulates aflatoxin biosynthesis in Aspergillus parasiticus. 1096 69

The Azotobacter vinelandii NIFL regulatory flavoprotein responds to the redox, energy and nitrogen status of the cell to inhibit transcriptional activation by the sigmaN-dependent enhancer binding protein, NIFA, via the formation of a NIFL-NIFA protein complex. The NIFA protein contains three domains: an N-terminal domain of unknown function; a central catalytic domain required to couple nucleotide hydrolysis to activation of the sigmaN-RNA polymerase holoenzyme; and a C-terminal DNA-binding domain. We report that truncated NIFA proteins that either lack the amino-terminal domain or contain only the isolated central domain remain responsive to inhibition by NIFL but, in contrast to native NIFA, continue to hydrolyse nucleotides when NIFL is present. We also report that NIFL is competent to inhibit the DNA-binding function of NIFA. Taken together, these results suggest that NIFL inhibits NIFA via a concerted mechanism in which DNA binding, catalytic activity and, potentially, interaction with the polymerase are controlled by NIFL in order to prevent transcriptional activation under detrimental environmental conditions.
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PMID:Concerted inhibition of the transcriptional activation functions of the enhancer-binding protein NIFA by the anti-activator NIFL. 1113 67

Here we describe the role of the Cladosporium fulvum nitrogen response factor 1 (Nrf1) gene in regulation of the expression of avirulence gene Avr9 and virulence on tomato. The Nrf1 gene, which was isolated by a polymerase chain reaction-based strategy, is predicted to encode a protein of 918 amino acid residues. The protein contains a putative zinc finger DNA-binding domain that shares 98% amino acid identity with the zinc finger of the major nitrogen regulatory proteins AREA and NIT2 of Aspergillus nidulans and Neurospora crassa, respectively. Functional equivalence of Nrf1 to areA was demonstrated by complementation of an A. nidulans areA loss-of-function mutant with Nrf1. Nrf1-deficient transformants of C. fulvum obtained by homologous recombination were unable to utilize nitrate and nitrite as a nitrogen source. In contrast to what was observed in the C. fulvum wild-type, the Avr9 gene was no longer induced under nitrogen-starvation conditions in Nrf1-deficient strains. On susceptible tomato plants, the Nrf1-deficient strains were as virulent as wild-type strains of C. fulvum, although the expression of the Avr9 gene was strongly reduced. In addition, Nrf1-deficient strains were still avirulent on tomato plants containing the functional Cf-9 resistance gene, indicating that in planta, apparently sufficient quantities of stable AVR9 elicitor are produced. Our results suggest that the NRF1 protein is a major regulator of the Avr9 gene.
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PMID:Expression of the Avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum is regulated by the global nitrogen response factor NRF1. 1127 29

Signal transduction by two-component regulatory systems involves phosphorylation of the receiver domain of a response regulator by the transmitter domain of the cognate histidine kinase. In the NtrBC system, phosphorylation of NtrC by NtrB results in transcriptional activation of nitrogen-regulated genes. We have used the yeast two-hybrid system to probe interactions between domains of the NtrB and NtrC proteins from Klebsiella pneumoniae. We constructed fusions from each of a series of proteins or protein domains to the activation and the DNA-binding domains of GAL4 and analysed expression of GAL1:lacZ and GAL1:HIS3 reporters in yeast. The DNA-binding domain of NtrC and the so-called sensor domain of NtrB appeared to provide the major determinants for dimerization of the fusion proteins. A strong and specific interaction was also shown between NtrB and NtrC, localized to the HN region of the NtrB transmitter module and to the NtrC receiver domain, whereas other domains of these proteins do not appear to contribute to the recognition specificity. The results presented here indicate that communication between two-component partners also involves protein-protein interactions that can be detected in vivo, suggesting that the yeast two-hybrid system is a powerful genetic tool for identifying functional partners of prokaryotic signal transduction pathways.
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PMID:Two-hybrid analysis of domain interactions involving NtrB and NtrC two-component regulators. 1129 84

Expression of nitrogen fixation genes in Rhodobacter capsulatus is repressed by ammonium at different regulatory levels including an NtrC-independent mechanism controlling NifA activity. In contrast to R. capsulatus NifA, heterologous NifA proteins of Klebsiella pneumoniae and Rhizobium meliloti, respectively, were not subjected to this posttranslational ammonium control in R. capsulatus. The characterization of ammonium-tolerant R. capsulatus NifA1 mutants indicated that the N-terminal domain of NifA was involved in posttranslational regulation. Analysis of a double mutant carrying amino acid substitutions in both the N-terminal domain and the C-terminal DNA-binding domain gave rise to the hypothesis that an interaction between these two domains might be involved in ammonium regulation of NifA activity. Western analysis demonstrated that both constitutively expressed wild-type and ammonium-tolerant NifA1 proteins exhibited high stability and accumulated to comparable levels in cells grown in the presence of ammonium excluding the possibility that proteolytic degradation was responsible for ammonium-dependent inactivation of NifA.
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PMID:Rhodobacter capsulatus nifA mutants mediating nif gene expression in the presence of ammonium. 1142 77

The tam A gene of Aspergillus nidulans encodes a 739-amino acid protein with similarity to Uga35p/Dal81p/DurLp of Saccharomyces cerevisiae. It has been proposed that TamA functions as a co-activator of AreA, the major nitrogen regulatory protein in A. nidulans. Because AreA functions as a transcriptional activator under nitrogen-limiting conditions, we investigated whether TamA was also present in the nucleus. We found that a GFP-TamA fusion protein was predominantly localised to the nucleus in the presence and absence of ammonium, and that AreA was not required for this distribution. As the predicted DNA-binding domain of TamA is not essential for function, we have used a number of approaches to further define functionally important regions. We have cloned the tamA gene of A. oryzae and compared its functional and sequence characteristics with those of A. nidulans tamA and S. cerevisiae UGA35/DAL81/DURL. The Aspergillus homologues are highly conserved and functionally interchangeable, whereas the S. cerevisiae gene does not complement a tamA mutant when expressed in A. nidulans. Uga35p/Dal81p/DurLp was also found to be unable to recruit AreA. The sequence changes in a number of tamA mutant alleles were determined, and altered versions of TamA were tested for tamA complementation and interaction with AreA. Changes in most regions of TamA appeared to destroy its function, suggesting that the overall conformation of the protein may be critical for its activity.
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PMID:Functional analysis of TamA, a coactivator of nitrogen-regulated gene expression in Aspergillus nidulans. 1145 83

In Bacillus subtilis, carbon catabolite control is mediated by the regulatory protein CcpA. In addition to loss of catabolite repression, ccpA mutants exhibit a severe growth defect. They are not able to grow with glucose and ammonium as single sources of carbon and nitrogen, respectively. Only ccpA mutant strains carrying either secondary suppressor mutations or that are affected in specific amino acids in the DNA-binding domain of CcpA grow on minimal media. We addressed the importance of DNA-binding by CcpA for both carbon catabolite repression and growth of a mutant strain. A strain specifically deleted for the N-terminal DNA-binding domain of CcpA was constructed. This deletion resulted in complete loss of catabolite repression of beta-xylosidase synthesis and prevented bacteria from growing on minimal media, suggesting that DNA-binding by CcpA is required for both carbon catabolite repression and efficient growth on minimal media.
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PMID:The Bacillus subtilis catabolite control protein CcpA exerts all its regulatory functions by DNA-binding. 1155 50

Two-component regulatory systems mediate most of the bacterial cells responses to a variety of signals. In Sinorhizobium meliloti, the FixL-FixJ couple controls the expression of the nitrogen fixation genes through the binding of the two-domains response regulator FixJ to the fixK and nifA promoters. Phosphorylation of the N-terminal regulatory domain activates the protein and releases the inhibition of the C-terminal DNA-binding domain that occurs in the unphosphorylated protein. Insights into the transition from the inactive to the active form are provided by the architecture of the unphosphorylated response regulator reported in this study. The relative position and orientation of the N and C-terminal domains were defined from the molecular envelope restored from small-angle X-ray scattering (SAXS) data. The involvement of the alpha4-beta5-alpha5 surface of the regulatory domain, the linker region and the C-terminal helix of the DNA-binding domain in the interdomain interface of unphosphorylated FixJ was supported by biochemical investigations. These results, together with the previously reported studies on the phosphorylated regulatory domain of FixJ, emphasize the role of the alpha4-beta5-alpha5 surface in mediating a flow of information in this response regulator. This first study by SAXS of proteins from two-component systems suggests that the method could be successfully applied to other members of this family and could be suitable for the study of multidomain proteins and protein-protein complexes regulated through molecular interfaces in the low micromolar range.
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PMID:Insights into signal transduction revealed by the low resolution structure of the FixJ response regulator. 1216 58

The NifA protein of Klebsiella pneumoniae is required for transcriptional activation of all nitrogen fixation (nif) operons except the regulatory nifLA genes. At these operons, NifA binds to an upstream activator sequence (UAS), with the consensus TGT-N(10)-ACA, via a C-terminal DNA-binding domain (CTD). Binding of the activator to this upstream enhancer-like sequence allows NifA to interact with RNA polymerase containing the alternative sigma factor, sigma(54). The isolated NifA CTD is monomeric and binds specifically to DNA in vitro as shown by DNase I footprinting. Heteronuclear 3D NMR experiments have been used to assign the signals from the protein backbone. Three alpha-helices have been identified, based on secondary chemical shifts and medium range Halpha(i)-NH(i)( + 1), and NH(i)-NH(i)( + 1) NOEs. On addition of DNA containing a half-site UAS, several changes are observed in the NMR spectra, allowing the identification of residues that are most likely to interact with DNA. These occur in the final two helices of the protein, directly confirming that DNA binding is mediated by a helix-turn-helix motif.
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PMID:Secondary structure and DNA binding by the C-terminal domain of the transcriptional activator NifA from Klebsiella pneumoniae. 1223 81


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