The DNA Binding Protein Rfg1 Is a Repressor of Filamentation inCandida albicans

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1503-1512 ◽  
Author(s):  
Roy A Khalaf ◽  
Richard S Zitomer
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Hyphal Growth ◽  
Dna Binding Protein ◽  
Homozygous Deletion ◽  
Wild Type ◽  
Pathogenic Yeast ◽  
Repression Complex ◽  
Type Gene ◽  
Repression Domain

AbstractWe have identified a repressor of hyphal growth in the pathogenic yeast Candida albicans. The gene was originally cloned in an attempt to characterize the homologue of the Saccharomyces cerevisiae Rox1, a repressor of hypoxic genes. Rox1 is an HMG-domain, DNA binding protein with a repression domain that recruits the Tup1/Ssn6 general repression complex to achieve repression. The C. albicans clone also encoded an HMG protein that was capable of repression of a hypoxic gene in a S. cerevisiae rox1 deletion strain. Gel retardation experiments using the purified HMG domain of this protein demonstrated that it was capable of binding specifically to a S. cerevisiae hypoxic operator DNA sequence. These data seemed to indicate that this gene encoded a hypoxic repressor. However, surprisingly, when a homozygous deletion was generated in C. albicans, the cells became constitutive for hyphal growth. This phenotype was rescued by the reintroduction of the wild-type gene on a plasmid, proving that the hyphal growth phenotype was due to the deletion and not a secondary mutation. Furthermore, oxygen repression of the hypoxic HEM13 gene was not affected by the deletion nor was this putative ROX1 gene regulated positively by oxygen as is the case for the S. cerevisiae gene. All these data indicate that this gene, now designated RFG1 for Repressor of Filamentous Growth, is a repressor of genes required for hyphal growth and not a hypoxic repressor.

scholarly journals φX174 Genome-Capsid Interactions Influence the Biophysical Properties of the Virion: Evidence for a Scaffolding-Like Function for the Genome during the Final Stages of Morphogenesis

2002 ◽  
Vol 76 (11) ◽  
pp. 5350-5356 ◽  
Author(s):  
Susan Hafenstein ◽  
Bentley A. Fane
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Coat Proteins ◽  
Wild Type ◽  
Migration Rates ◽  
Protein Dna Interactions ◽  
Inner Surface ◽  
Native Gel ◽  
Host Cell Recognition

ABSTRACT During the final stages of φX174 morphogenesis, there is an 8.5-Å radial collapse of coat proteins around the packaged genome, which is tethered to the capsid's inner surface by the DNA-binding protein. Two approaches were taken to determine whether protein-DNA interactions affect the properties of the mature virion and thus the final stages of morphogenesis. In the first approach, genome-capsid associations were altered with mutant DNA-binding proteins. The resulting particles differed from the wild-type virion in density, native gel migration, and host cell recognition. Differences in native gel migration were especially pronounced. However, no differences in protein stoichiometries were detected. An extragenic second-site suppressor of the mutant DNA-binding protein restores all assayed properties to near wild-type values. In the second approach, φX174 was packaged with foreign, single-stranded, covalently closed, circular DNA molecules identical in length to the φX174 genome. The resulting particles exhibited native gel migration rates that significantly differed from the wild type. The results of these experiments suggest that the structure of the genome and/or its association with the capsid's inner surface may perform a scaffolding-like function during the procapsid-to- virion transition.

scholarly journals Characterization of the Pathway-Specific Positive Transcriptional Regulator for Actinorhodin Biosynthesis inStreptomyces coelicolor A3(2) as a DNA-Binding Protein

1999 ◽  
Vol 181 (22) ◽  
pp. 6958-6968 ◽  
Author(s):  
Paloma Arias ◽  
Miguel A. Fernández-Moreno ◽  
Francisco Malpartida
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Regulatory Protein ◽  
Dna Binding Protein ◽  
Binding Activity ◽  
Wild Type ◽  
Biosynthetic Genes ◽  
Dna Binding Activity ◽  
Orf4 Protein

ABSTRACT The ActII-ORF4 protein has been characterized as a DNA-binding protein that positively regulates the transcription of the actinorhodin biosynthetic genes. The target regions for the ActII-ORF4 protein were located within the act cluster. These regions, at high copy number, generate a nonproducer strain by in vivo titration of the regulator. The mutant phenotype could be made to revert with extra copies of the wild-type actII-ORF4 gene but not with theactII-ORF4-177 mutant. His-tagged recombinant wild-type ActII-ORF4 and mutant ActII-ORF4-177 proteins were purified fromEscherichia coli cultures; both showed specific DNA-binding activity for the actVI-ORF1–ORFA andactIII-actI intergenic regions. DNase I footprinting assays clearly located the DNA-binding sites within the −35 regions of the corresponding promoters, showing the consensus sequence 5′-TCGAG-3′. Although both gene products (wild-type and mutant ActII-ORF4) showed DNA-binding activity, only the wild-type gene was capable of activating transcription of the actgenes; thus, two basic functions can be differentiated within the regulatory protein: a specific DNA-binding activity and a transcriptional activation of the act biosynthetic genes.

scholarly journals A Novel DNA-Binding Protein Plays an Important Role in Helicobacter pylori Stress Tolerance and Survival in the Host

2014 ◽  
Vol 197 (5) ◽  
pp. 973-982 ◽  
Author(s):  
Ge Wang ◽  
Robert J. Maier
Keyword(s):  
Oxidative Stress ◽  
Helicobacter Pylori ◽  
Dna Binding ◽  
Stress Tolerance ◽  
Mutant Strain ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Wild Type ◽  
Content Type ◽  
H Pylori

The gastric pathogenHelicobacter pylorimust combat chronic acid and oxidative stress. It does so via many mechanisms, including macromolecule repair and gene regulation. Mitomycin C-sensitive clones from a transposon mutagenesis library were screened. One sensitive strain contained the insertion element at the locus ofhp119, a hypothetical gene. No homologous gene exists in any (non-H. pylori) organism. Nevertheless, the predicted protein has some features characteristic of histone-like proteins, and we showed that purified HP119 protein is a DNA-binding protein. A Δhp119strain was markedly more sensitive (viability loss) to acid or to air exposure, and these phenotypes were restored to wild-type (WT) attributes upon complementation of the mutant with the wild-type version ofhp119at a separate chromosomal locus. The mutant strain was approximately10-fold more sensitive to macrophage-mediated killing than the parent or the complemented strain. Of 12 mice inoculated with the wild type, all containedH. pylori, whereas 5 of 12 mice contained the mutant strain; the mean colonization numbers were 158-fold less for the mutant strain. A proteomic (two-dimensional PAGE with mass spectrometric analysis) comparison between the Δhp119mutant and the WT strain under oxidative stress conditions revealed a number of important antioxidant protein differences; SodB, Tpx, TrxR, and NapA, as well as the peptidoglycan deacetylase PgdA, were significantly less expressed in the Δhp119mutant than in the WT strain. This study identified HP119 as a putative histone-like DNA-binding protein and showed that it plays an important role inHelicobacter pyloristress tolerance and survival in the host.

scholarly journals Multiple Modes of Interaction between the Methylated DNA Binding Protein MeCP2 and Chromatin

2006 ◽  
Vol 27 (3) ◽  
pp. 864-877 ◽  
Author(s):  
Tatiana Nikitina ◽  
Xi Shi ◽  
Rajarshi P. Ghosh ◽  
Rachel A. Horowitz-Scherer ◽  
Jeffrey C. Hansen ◽  
...  
Keyword(s):  
Dna Binding ◽  
Conformational Changes ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Chromatin Interaction ◽  
Wild Type ◽  
Mobility Shift ◽  
Methylated Dna ◽  
Neurodevelopmental Disease ◽  
Protein Mecp2

ABSTRACT Mutations of the methylated DNA binding protein MeCP2, a multifunctional protein that is thought to transmit epigenetic information encoded as methylated CpG dinucleotides to the transcriptional machinery, give rise to the debilitating neurodevelopmental disease Rett syndrome (RTT). In this in vitro study, the methylation-dependent and -independent interactions of wild-type and mutant human MeCP2 with defined DNA and chromatin substrates were investigated. A combination of electrophoretic mobility shift assays and visualization by electron microscopy made it possible to understand the different conformational changes underlying the gel shifts. MeCP2 is shown to have, in addition to its well-established methylated DNA binding domain, a methylation-independent DNA binding site (or sites) in the first 294 residues, while the C-terminal portion of MeCP2 (residues 295 to 486) contains one or more essential chromatin interaction regions. All of the RTT-inducing mutants tested were quantitatively bound to chromatin under our conditions, but those that tend to be associated with the more severe RTT symptoms failed to induce the extensive compaction observed with wild-type MeCP2. Two modes of MeCP2-driven compaction were observed, one promoting nucleosome clustering and the other forming DNA-MeCP2-DNA complexes. MeCP2 binding to DNA and chromatin involves a number of different molecular interactions, some of which result in compaction and oligomerization. The multifunctional roles of MeCP2 may be reflected in these different interactions.

scholarly journals A DNA-binding protein tunes septum placement duringBacillus subtilissporulation

2018 ◽  
Author(s):  
Emily E. Brown ◽  
Allyssa K. Miller ◽  
Inna V. Krieger ◽  
Ryan M. Otto ◽  
James C. Sacchettini ◽  
...  
Keyword(s):  
Cell Division ◽  
Dna Binding ◽  
Vegetative Growth ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Fine Tuning ◽  
Wild Type ◽  
Circular Chromosome ◽  
Spore Form ◽  
Dna Motifs

AbstractBacillus subtilisis a soil bacterium capable of differentiating into a spore form resistant to desiccation, UV radiation, and heat. Early in spore development the cell possesses two copies of a circular chromosome, anchored to opposite cell poles via DNA proximal to the origin of replication (oriC). As sporulation progresses an FtsZ ring (Z-ring) assembles close to one pole and directs septation over one chromosome. The polar division generates two cell compartments with differing chromosomal contents. The smaller “forespore” compartment initially contains only 25–30% of one chromosome and this transient genetic asymmetry is required for differentiation. At the population level, the timely assembly of polar Z-rings and the precise capture of the chromosome in the forespore both require RefZ, a DNA-binding protein synthesized early in sporulation. To mediate precise capture of the chromosome RefZ must bind to specific DNA motifs (RBMs) that are localized near the poles around the time of septation, suggesting RefZ binds to theRBMsto affect positioning of the septum relative to the chromosome. RefZ’s mechanism of action is unknown, however, cells artificially induced to express RefZ during vegetative growth cannot assemble Z-rings or divide, leading to the hypothesis that RefZ-RBM complexes mediate precise chromosome capture by modulating FtsZ function. To investigate this possibility, we isolated 10 RefZ loss-of-function (rLOF) variants unable to inhibit cell division when expressed during vegetative growth, yet were still capable of bindingRBM-containing DNA. Sporulating cells expressing the rLOF variants in place of wild-type RefZ phenocopy a ΔrefZmutant, suggesting that RefZ mediates chromosome capture through an FtsZ-dependent mechanism. To better understand the molecular basis of RefZ’s activity, the crystal structure of RefZ was solved and wild-type RefZ and the rLOF variants were further characterized. Our data suggest that RefZ’s oligomerization state and specificity for theRBMsare critical determinants influencing RefZ’s ability to affect FtsZ dynamicsin vivo. We propose that RBM-bound RefZ complexes function as a developmentally regulated nucleoid occlusion system for fine-tuning the position of the septum relative to the chromosome during sporulation.Author SummaryThe Gram-positive bacteriumB. subtiliscan differentiate into a dormant cell type called a spore. Early in sporulation the cell divides near one pole, generating two compartments: a larger mother cell and a smaller forespore (future spore). Only approximately 30 percent of one chromosome is initially captured in the forespore compartment at the time of division and this genetic asymmetry is critical for sporulation to progress. Precise chromosome capture requires RefZ, a sporulation protein that binds to specific DNA motifs (RBMs) positioned at the pole near the site of cell division. How RefZ functions at the molecular level is not fully understood. Here we show that RefZ-RBMcomplexes facilitate chromosome capture by acting through the major cell division protein FtsZ.

scholarly journals A DNA-Binding Protein Tunes Septum Placement duringBacillus subtilisSporulation

2019 ◽  
Vol 201 (16) ◽  
Author(s):  
Emily E. Brown ◽  
Allyssa K. Miller ◽  
Inna V. Krieger ◽  
Ryan M. Otto ◽  
James C. Sacchettini ◽  
...  
Keyword(s):  
Cell Division ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Fine Tuning ◽  
Wild Type ◽  
Circular Chromosome ◽  
Spore Form ◽  
Content Type ◽  
Dna Motifs

ABSTRACTBacillus subtilisis a bacterium capable of differentiating into a spore form more resistant to environmental stress. Early in sporulation, each cell possesses two copies of a circular chromosome. A polar FtsZ ring (Z ring) directs septation over one of the chromosomes, generating two cell compartments. The smaller “forespore” compartment initially contains only 25 to 30% of one chromosome, and this transient genetic asymmetry is required for differentiation. Timely assembly of polar Z rings and precise capture of the chromosome in the forespore both require the DNA-binding protein RefZ. To mediate its role in chromosome capture, RefZ must bind to specific DNA motifs (RBMs) that localize near the poles at the time of septation. Cells artificially induced to express RefZ during vegetative growth cannot assemble Z rings, an effect that also requires DNA binding. We hypothesized that RefZ-RBMcomplexes mediate precise chromosome capture by modulating FtsZ function. To investigate, we isolated 10 RefZ loss-of-function (rLOF) variants unable to inhibit cell division yet still capable of bindingRBMs. Sporulating cells expressing the rLOF variants in place of wild-type RefZ phenocopied a ΔrefZmutant, suggesting that RefZ acts through an FtsZ-dependent mechanism. The crystal structure of RefZ was solved, and wild-type RefZ and the rLOF variants were further characterized. Our data suggest that RefZ’s oligomerization state and specificity for theRBMs are critical determinants influencing RefZ’s ability to affect FtsZ dynamics. We propose thatRBM-bound RefZ complexes function as a developmentally regulated nucleoid occlusion system for fine-tuning the position of the septum relative to the chromosome during sporulation.IMPORTANCEThe bacterial nucleoid forms a large, highly organized structure. Thus, in addition to storing the genetic code, the nucleoid harbors positional information that can be leveraged by DNA-binding proteins to spatially constrain cellular activities. DuringB. subtilissporulation, the nucleoid undergoes reorganization, and the cell division protein FtsZ assembles polarly to direct septation over one chromosome. The TetR family protein RefZ binds DNA motifs (RBMs) localized near the poles at the time of division and is required for both timely FtsZ assembly and precise capture of DNA in the future spore compartment. Our data suggest that RefZ exploits nucleoid organization by associating with polarly localizedRBMs to modulate the positioning of FtsZ relative to the chromosome during sporulation.

scholarly journals Combinatorial Repression of the Hypoxic Genes of Saccharomyces cerevisiae by DNA Binding Proteins Rox1 and Mot3

2005 ◽  
Vol 4 (4) ◽  
pp. 649-660 ◽  
Author(s):  
Lee G. Klinkenberg ◽  
Thomas A. Mennella ◽  
Katharina Luetkenhaus ◽  
Richard S. Zitomer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Aerobic Growth ◽  
Repression Complex ◽  
Combinatorial Interactions

ABSTRACT The hypoxic genes of Saccharomyces cerevisiae are transcriptionally repressed during aerobic growth through recruitment of the Ssn6/Tup1 general repression complex by the DNA binding protein Rox1. A second DNA binding protein Mot3 enhances repression of some hypoxic genes. Previous studies characterized the role of Mot3 at the hypoxic ANB1 gene as promoting synergy among one Mot3 site and two Rox1 sites comprising operator A of that gene. Here we studied the role of Mot3 in enhancing repression by Rox1 at another hypoxic gene, HEM13, which is less strongly regulated than ANB1 and has a very different arrangement of Rox1 and Mot3 binding sites. By assessing the effects of deleting Rox1 and Mot3 sites individually and in combination, we found that the major repression of HEM13 occurred through three Mot3 sites closely spaced with a single Rox1 site. While the Mot3 sites functioned additively, they enhanced repression by the single Rox1 site, and the presence of Rox1 enhanced the additive effects of the Mot3 sites. In addition, using a Rox1-Ssn6 fusion protein, we demonstrated that Mot3 enhances Rox1 repression through helping recruit the Ssn6/Tup1 complex. Chromatin immunoprecipitation assays indicated that Rox1 stabilized Mot3 binding to DNA. Integrating these results, we were able to devise a set of rules that govern the combinatorial interactions between Rox1 and Mot3 to achieve differential repression.

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