DNA-binding Transcriptional Regulators Research Articles

In Vivo and In Vitro Characterization of the ARR11 Response Regulator Implicated in the His-to-Asp Phosphorelay Signal Transduction in Arabidopsis thaliana

In Arabidopsis thaliana, Histidine-to-Aspartate (His--> Asp) phosphorelay is a paradigm of a signaling system that is considered to be involved in response to plant hormones, including ethylene and cytokinin. In the current framework of His-->Asp phosphorelay in this higher plant, the type-B ARR (response regulator) family members appear to act as DNA-binding transcriptional regulators. Although Arabidopsis thaliana has 11 type-B ARR family members, except for ARR1 and ARR2, no biological information is available with regard to others. As the main objective of this study, we characterized another example, ARR11, in terms of not only its in vitro biochemical properties, but also its biological activity in plants. In plants, the ARR11 gene was expressed predominantly in roots. In vitro, ARR11 showed the ability to acquire a phosphoryl group from a histidine-containing phosphotransfer intermediate (AHP), and also it showed the ability to recognize a specific nucleotide sequence, GGATT. These in vitro results supported the view that ARR11 is indeed a DNA-binding transcription factor, the ability of which is most likely modulated by phosphorylation in its receiver domain. In vivo, when a C-terminal DNA-binding domain lacking the N-terminal phospho-accepting (or receiver) domain was aberrantly expressed, the resulting transgenic plants showed characteristic anomalies during development of apical parts. The observed anomalies included "unusual proliferation of tissues in cotyledons" and "outgrowth of adventitious shoots near cotyledons". These results with regard to the functions of ARR11 are mainly discussed in comparison with those of the previously characterized type-B response regulators.

Genome biology of a model bacterium, Bacillus subtilis

Bacillus subtilis has served as a model bacterium in biochemical, genetic and molecular biological studies. The complete genome sequence of B. subtilis was determined by an international consortium, and a collection of knockout mutants of genes whose specific functions were not known was also generated by an international effort. As a result, 2810 knockout and 135 IPTG-dependent mutants have been constructed. It has been estimated that 275 genes, including 25 genes of unknown function, are essential for B. subtilis growth in rich medium at a moderate temperature in aerobic conditions. The B. subtilis genome has 17 sigma factors and about 250 DNA binding transcriptional regulators. In order to develop a comprehensive picture of the regulatory network of gene expression in B. subtilis, a systematic effort to collect expression profiles of wild type cells and knockout mutants of regulator genes is in progress. Mutant resources and genome-wide functional data sets will further assist in elucidation of a complete picture of the functional network involving all B. subtilis gene products.

DNA binding and dimerization specificity and potential targets for the TCP protein family.

The TCP domain is a plant-specific DNA binding domain found in proteins from a diverse array of species, including the cycloidea (cyc) and teosinte branched1 (tb1) gene products and the PCF1 and PCF2 proteins. To understand the role in transcriptional regulation of proteins with this domain, we have analysed the DNA binding and dimerization specificity of the TCP protein family using rice PCF proteins, and further evaluated potential targets for the TCP protein. The seven PCF members including five newly isolated proteins, were able to be grouped into two classes, I and II, based on sequence similarity in the TCP domain. Random binding site selection experiments and electrophoretic mobility shift assays (EMSAs) revealed the consensus DNA binding sequences of these two classes to be distinct but overlapping; GGNCCCAC for class I and GTGGNCCC for class II. The TB1 protein from maize, which belongs to class II, had the same specificity as the rice class II proteins, suggesting the conservation of binding specificity between TCP domains from different species. The yeast 2-hybrid assay and EMSA revealed that these proteins tend to form a homodimer or a heterodimer between members of the same class. We searched predicted 5' flanking sequences of Arabidopsis genes for the consensus binding sequences and found that the consensus sites are distributed in the genome at a considerably lower frequency. We further analysed eight promoters containing the class I consensus TCP sites. The transcriptional activities of six promoters were decreased by a mutation of the TCP binding site, which is consistent with the observation that the class I TCP site can confer transactivation function on a heterologous promoter. These results suggest that the two classes of TCP protein are distinct in DNA binding specificity and transcriptional regulation.

The nuclear receptor Ftz-F1 and homeodomain protein Ftz interact through evolutionarily conserved protein domains.

The Drosophila homeodomain protein Fushi Tarazu (Ftz) and its partner, the orphan receptor Ftz-F1, are members of two distinct families of DNA binding transcriptional regulators. Ftz and Ftz-F1 form a novel partnership in vivo as a Hox/orphan receptor heterodimer. Here we show that the murine Ftz-F1 ortholog SF-1 functionally substitutes for Ftz-F1 in vivo, rescuing the defects of ftz-f1 mutants. This finding identified evolutionarily conserved domains of Ftz-F1 as critical for activity of this receptor in vivo. These domains function, at least in part, by mediating direct protein interactions with Ftz. The Ftz-F1 DNA binding domain interacts strongly with Ftz and dramatically facilitates the binding of Ftz to target DNA. This interaction is augmented by a second interaction between the AF-2 domain of Ftz-F1 and the N-terminus of Ftz via an LRALL sequence in Ftz that is reminiscent of LXXLL motifs in nuclear receptor coactivators. We propose that Ftz-F1 serves as a cofactor for Ftz by facilitating the selection of target sites in the genome that contain Ftz/Ftz-F1 composite binding sites. Ftz, on the other hand, influences Ftz-F1 activity by interacting with its AF-2 domain in a manner that mimics a nuclear receptor coactivator.

RegulonDB (version 3.2): transcriptional regulation and operon organization in Escherichia coli K-12.

RegulonDB is a database on mechanisms of transcription regulation and operon organization in Escherichia coli K-12. The current version has considerably increased numbers of regulatory elements such as promoters, binding sites and terminators. The complete repertoire of known and predicted DNA-binding transcriptional regulators can be considered to be included in this version. The database now distinguishes different allosteric conformations of regulatory proteins indicating the one active in binding and regulating the different promoters. A new set of operon predictions has been incorporated. The relational design has been modified accordingly. Furthermore, a major improvement is a graphic display enabling browsing of the database with a Java-based graphic user interface with three zoom-levels connected to properties of each chromosomal element. The purpose of these modifications is to make RegulonDB a useful tool and control set for transcriptome experiments. RegulonDB can be accessed on the web at the URL: http://www.cifn.unam.mx/Computational_Biology/++ +regulondb/

Molecular analysis of the mannitol operon of Clostridium acetobutylicum encoding a phosphotransferase system and a putative PTS-modulated regulator.

Clostridium acetobutylicum DSM 792 accumulates and phosphorylates mannitol via a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). PEP-dependent mannitol phosphorylation by extracts of cells grown on mannitol required both soluble and membrane fractions. Neither the soluble nor the membrane fraction could be complemented by the opposite fraction prepared from glucose-grown cells, indicating that the mannitol-specific PTS consists of both a soluble (IIA) and a membrane-bound (IICB) component. The mannitol (mtl) operon of C. acetobutylicum DSM 792 comprises four genes in the order mtlARFD. Sequence analysis of deduced protein products indicated that the mtlA and mtlF genes respectively encode the IICB and IIA components of the mannitol PTS, which is a member of the fructose-mannitol (Fru) family. The mtlD gene product is a mannitol-1-phosphate dehydrogenase, while mtlR encodes a putative transcriptional regulator. MtlR contains two PTS regulatory domains (PRDs), which have been found in a number of DNA-binding transcriptional regulators and in transcriptional antiterminators of the Escherichia coli BglG family. Also, near the C-terminus is a well-conserved signature motif characteristic of members of the IIA(Fru)/IIA(Mtl)/IIA(Ntr) PTS protein family. These regions are probably the sites of PTS-dependent phosphorylation to regulate the activity of the protein. A helix-turn-helix DNA-binding motif was not found in MtlR. Transcriptional analysis of the mtl genes by Northern blotting indicated that the genes were transcribed as a polycistronic operon, expression of which was induced by mannitol and repressed by glucose. Primer extension experiments identified a transcriptional start point 42 bp upstream of the mtlA start codon. Two catabolite-responsive elements (CREs), one of which overlapped the putative -35 region of the promoter, were located within the 100 bp upstream of the start codon. These sequences may be involved in regulation of expression of the operon.

A zinc transporter in Synechocystis PCC 6803 and DNA microtip analysis of gene expression in response to changes in zinc concentration

Zinc is an abundant transition metal in the biosphere. Because of its Lewis acidity, flexible cordination geometry, rapid ligand exchange property and absence of redox activity, Zn is used as a metal of choice in many biological reactions. In addition, Zn-binding Zn finger protein form an important class of DNA-binding transcriptional regulators. A number of Zn transporter systems have been identified in yeast, human and plants. An ABC-type Zn-permease and a Zn-binding repressor protein have been identified in E. coli. The complete genome sequence of Synechocystis PCC 6803 revealed that the slr2043-slr2044-slr2045 cluster of ORFs are homologous to the mntCAB (encoding a Mn-permease). Inactivation of each of these ORFs in this study resulted in mutant strains that grow poorly in the absence of added Zn in the growth medium. Analysis of these mutant strains has established that this operon encodes a Zn-uptake transporter. Zn is toxic to the cells and Synechocystis possesses Zn exporter encoded by slr0798 to keep the intracellular Zn concentration at low level. DNA microarray analysis revealed that the expression of many genes is up-regulated or down-regulated in response to the increase of external Zn concentration. We shall enlist these genes.

A novel ability of Smad3 to regulate proteasomal degradation of a Cas family member HEF1.

Smad3 is a key signal transducer of transforming growth factor-ss (TGF-ss) and activin, and is known to be a DNA-binding transcriptional regulator. Here we report a novel property of Smad3 in regulating the proteasomal degradation of the human enhancer of filamentation 1 (HEF1), which is a member of the Cas family of cytoplasmic docking proteins. Our studies revealed that Smad3 interacts with HEF1 and triggers the proteasomal degradation of HEF1 in overexpression systems. In addition, TGF-ss stimulation induces rapid proteasomal degradation of endogenous HEF1 in different TGF-ss-responsive cell lines. Interestingly, the degradation of HEF1 protein in epithelial cells is followed closely by an increase in HEF1 mRNA, resulting in a time-dependent increase in HEF1 protein level in TGF-ss-treated cells. Furthermore, we observed that an elevated HEF1 protein level inhibits TGF-ss-induced Smad3-mediated gene responses. These data provide the first evidence for a novel cytoplasmic activity of Smad3 in regulating proteasomal degradation of HEF1 and also suggest a role for HEF1 in a negative feedback mechanism of the TGF-ss signaling pathway.

The abaA homologue of Penicillium marneffei participates in two developmental programmes: conidiation and dimorphic growth.

Penicillium marneffei is the only known species of its genus that is dimorphic. At 25 degrees C, P. marneffei exhibits true filamentous growth and undergoes asexual development producing spores borne on complex structures called conidiophores. At 37 degrees C, P. marneffei undergoes a dimorphic transition to produce uninucleate yeast cells that divide by fission. We have cloned a homologue of the Aspergillus nidulans abaA gene encoding an ATTS/TEA DNA-binding domain transcriptional regulator and shown that it is involved in both these developmental programs. Targeted deletion of abaA blocks asexual development at 25 degrees C before spore production, resulting in aberrant conidiophores with reiterated terminal cells. At 37 degrees C, the abaA deletion strain fails to switch correctly from multinucleate filamentous to uninucleate yeast cells. Both the transitional hyphal cells, which produce the yeast cells, and the yeast cells themselves contain multiple nuclei. Expression of the abaA gene is activated during both conidiation and the hyphal-yeast switch. Interestingly, the abaA gene of the filamentous monomorphic fungus A. nidulans can complement both conidiation and dimorphic switching defects in the P. marneffei abaA mutant. In addition, ectopic overexpression of abaA results in anucleate yeast cells and multinucleate vegetative filamentous cells. These data suggest that abaA regulates cell cycle events and morphogenesis in two distinct developmental programmes.

Human Slug is a repressor that localizes to sites of active transcription.

Snail/Slug family proteins have been identified in diverse species of both vertebrates and invertebrates. The proteins contain four to six zinc fingers and function as DNA-binding transcriptional regulators. Various members of the family have been demonstrated to regulate cell movement, neural cell fate, left-right asymmetry, cell cycle, and apoptosis. However, the molecular mechanisms of how these regulators function and the target genes involved are largely unknown. In this report, we demonstrate that human Slug (hSlug) is a repressor and modulates both activator-dependent and basal transcription. The repression depends on the C-terminal DNA-binding zinc fingers and on a separable repression domain located in the N terminus. This domain may recruit histone deacetylases to modify the chromatin and effect repression. Protein localization study demonstrates that hSlug is present in discrete foci in the nucleus. This subnuclear pattern does not colocalize with the PML foci or the coiled bodies. Instead, the hSlug foci overlap extensively with areas of the SC-35 staining, some of which have been suggested to be sites of active splicing or transcription. These results lead us to postulate that hSlug localizes to target promoters, where activation occurs, to repress basal and activator-mediated transcription.

The repertoire of DNA-binding transcriptional regulators in Escherichia coli K-12.

Using a combination of several approaches we estimated and characterized a total of 314 regulatory DNA-binding proteins in Escherichia coli, which might represent its minimal set of transcription factors. The collection is comprised of 35% activators, 43% repressors and 22% dual regulators. Within many regulatory protein families, the members are homogeneous in their regulatory roles, physiology of regulated genes, regulatory function, length and genome position, showing that these families have evolved homogeneously in prokaryotes, particularly in E.coli. This work describes a full characterization of the repertoire of regulatory interactions in a whole living cell. This repertoire should contribute to the interpretation of global gene expression profiles in both prokaryotes and eukaryotes.

In vitro analysis of the butyrolactone autoregulator receptor protein (FarA) of Streptomyces lavendulae FRI-5 reveals that FarA acts as a DNA-binding transcriptional regulator that controls its own synthesis.

FarA of Streptomyces lavendulae FRI-5 is a specific receptor protein for IM-2, a butyrolactone autoregulator that controls the production of a blue pigment and the nucleoside antibiotics showdomycin and minimycin. Gel shift assays demonstrated that FarA binds to the farA upstream region and that this binding is abolished in the presence of IM-2. The FarA binding sequence was localized by DNase I footprinting to a 28-bp sequence located approximately 70 bp upstream of the farA translational start site. High-resolution S1 nuclease mapping of farA transcripts revealed a putative transcription start site, located at an A residue positioned 64 bp upstream from the farA translation start codon and 4 bp downstream from an Escherichia coli sigma(70)-like -10 recognition region. The FarA-binding sequence overlaps this -10 region and contains the farA transcription initiation site, suggesting that FarA acts as a repressor that, in the absence of IM-2, represses transcription of farA.

A Double Interaction Screen identifies positive and negative ftz gene regulators and Ftz-interacting proteins

Regulatory genes directing embryonic development are expressed in complex patterns. The Drosophila homeobox gene fushi tarazu ( ftz) is expressed in a striped pattern that is controlled by several discrete and large cis- regulatory elements. One key cis-element is the ftz proximal enhancer which is required for stripe establishment and which mediates autoregulation by direct binding of Ftz protein. To identify the trans-acting factors that regulate ftz expression and autoregulation, we developed a modified yeast two hybrid screen, the Double Interaction Screen (DIS). The DIS was designed to isolate both DNA binding transcriptional regulators that interact with the proximal enhancer and proteins that interact with Ftz itself when it is bound to the enhancer. The screen identified two candidate Ftz protein cofactors as well as activators and repressors of ftz transcription that bind directly to the enhancer. One of these (Tramtrack Ttk) was previously shown to bind to at least five sites in the proximal enhancer; genetic studies suggested that Ttk acts as a repressor of ftz in the embryo. Here we show that, in yeast cells, Ttk protein strongly activates transcription, suggesting that yeast may be missing a necessary co-repressor which is present in Drosophila embryos. Further, we have characterized the activity of a second candidate ftz repressor isolated in the screen – the product of the pair-rule gene sloppy paired – a member of the forkhead family. We show that Slp1 is a DNA binding protein. We have identified a high affinity binding site for Slp1 in the ftz proximal enhancer. Slp1 represses transcription via this binding site in yeast cells, consistent with its role as a direct repressor of ftz stripes in interstripe regions during late stages of embryogenesis. The DIS should be a generally useful method to identify DNA binding transcriptional regulators and protein partners of previously characterized DNA binding proteins.

DREAM is a Ca2+-regulated transcriptional repressor.

Fluxes in amounts of intracellular calcium ions are important determinants of gene expression. So far, Ca2+-regulated kinases and phosphatases have been implicated in changing the phosphorylation status of key transcription factors and thereby modulating their function. In addition, direct effectors of Ca2+-induced gene expression have been suggested to exist in the nucleus, although no such effectors have been identified yet. Expression of the human prodynorphin gene, which is involved in memory acquisition and pain, is regulated through its downstream regulatory element (DRE) sequence, which acts as a location-dependent gene silencer. Here we isolate a new transcriptional repressor, DRE-antagonist modulator (DREAM), which specifically binds to the DRE. DREAM contains four Ca2+-binding domains of the EF-hand type. Upon stimulation by Ca2+, DREAM's ability to bind to the DRE and its repressor function are prevented. Mutation of the EF-hands abolishes the response of DREAM to Ca2+. In addition to the prodynorphin promoter, DREAM represses transcription from the early response gene c-fos. Thus, DREAM represents the first known Ca2+-binding protein to function as a DNA-binding transcriptional regulator.

The DNA binding protein Tfx from Methanobacterium thermoautotrophicum: structure, DNA binding properties and transcriptional regulation.

In Methanobacterium thermoautotrophicum, the fmdECB operon encoding the molybdenum formyl-methanofuran dehydrogenase is directly preceded by an open reading frame tfx predicted to encode a DNA binding protein. The 16.1 kDa protein has an N-terminal basic domain with a helix-turn-helix motif for DNA binding and a C-terminal acidic domain possibly for transcriptional activation. We report here on the DNA binding properties of the Tfx protein heterologously overproduced in Escherichia coli. Tfx was found to bind specifically to a DNA sequence downstream of the promoter of the fmdECB operon, as shown by electrophoretic mobility shift assays and DNase I footprint analysis. Northern blot hybridizations revealed that transcription of tfx is repressed during the growth of M. thermoautotrophicum in the presence of tung-state. Based on its structure and properties, the DNA binding protein Tfx is proposed to be a transcriptional regulator composed of a basic DNA binding domain and an acidic activation domain.

A dual-signaling mechanism mediated by the ArcB hybrid sensor kinase containing the histidine-containing phosphotransfer domain in Escherichia coli.

The two components ArcB and ArcA play a crucial role in the signal transduction implicated in the complex transcriptional regulatory network that allows Escherichia coli to sense various respiratory growth conditions. ArcB is a hybrid sensor kinase having multiple phosphorylation sites in its primary amino acid sequence, including a transmitter, a receiver, and a histidine-containing phosphotransfer (HPt) domain. ArcA is a DNA-binding transcriptional regulator with a receiver domain. Results of recent in vitro studies revealed multistep His-to-Asp phosphotransfer circuitry in the ArcB-ArcA signaling system. For this report we conducted a series of in vivo experiments using a set of crucial ArcB mutants to evaluate the regulation of the sdh operon. The results suggested that the phosphorylated His-717 site in the HPt domain of ArcB is essential for anaerobic repression of sdh. Nonetheless, the ArcB mutant lacking this crucial His-717 site does not necessarily exhibit a null phenotype with respect to ArcB-ArcA signaling. The HPt mutant appears to maintain an ability to signal ArcA, particularly under aerobic conditions, which results in a significant repression of sdh. Based on these and other in vivo results, we propose a model in which ArcB functions in its own right as a dual-signaling sensor that is capable of propagating two types of stimuli through two distinct phosphotransfer pathways.

Multiple phosphorylation of SacY, a Bacillus subtilis transcriptional antiterminator negatively controlled by the phosphotransferase system.

The Bacillus subtilis SacY transcriptional antiterminator is a regulator involved in sucrose-promoted induction of the sacB gene. SacY activity is negatively controlled by enzyme I and HPr, the general energy coupling proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and by SacX, a membranal protein homologous to SacP, the B. subtilis sucrose-specific PTS-permease. Previous studies suggested that the negative control exerted by the PTS on bacterial antiterminators of the SacY family involves phosphoenolpyruvate-dependent phosphorylation by the sugar-specific PTS-permeases. However, data reported herein show direct phosphorylation of SacY by HPr(His approximately P) with no requirement for SacX. Experiments were carried out to determine the phosphorylatable residues in SacY. In silico analyses of SacY and its homologues revealed the modular structure of these proteins as well as four conserved histidines within two homologous domains (here designated P1 and P2), present in 14 distinct mRNA- and DNA-binding bacterial transcriptional regulators. Single or multiple substitutions of these histidyl residues were introduced in SacY by site-directed mutagenesis, and their effects on phosphorylation and antitermination activity were examined. In vitro phosphorylation experiments showed that SacY was phosphorylated on three of the conserved histidines. Nevertheless, in vivo studies using cells bearing a sacB'-lacZ reporter fusion, as well as SacY mutants lacking the phosphorylatable histidyls, revealed that only His-99 is directly involved in regulation of SacY antitermination activity.

Expansion of the Hox gene family and the evolution of chordates.

Homeobox genes encode DNA-binding transcription regulators that participate in the formation of embryonic pattern or contribute to cell-type specificity during metazoan development. Homeobox genes that regulate axial patterning and segmental identity (Hox/HOM genes) share a conserved clustered genomic organization. Mammals have four clusters that have likely arisen from the duplication of a single ancestral cluster. The number of Hox-type genes in other deuterostomes was estimated by using a polymerase chain reaction sampling method. Increased Hox gene complements are associated with the appearance of chordate and vertebrate characters. Our data suggest the presence of one Hox cluster in the acorn worm, a hemichordate; two Hox clusters in amphioxus, a cephalochordate; and three in the lamprey, a primitive vertebrate.

Overproduction, solubilization, purification and DNA-binding properties of AmpR from Citrobacter freundii.

AmpR belongs to the LysR family of prokaryotic DNA-binding transcriptional regulators and controls induction of the enterobacterial ampC beta-lactamase gene. The ampR gene of Citrobacter freundii was deregulated by employing the polymerase chain reaction to introduce an efficient ribosome-binding sequence and suitable restriction enzyme sites for cloning into a chemically inducible tac-promoter expression vector. When induced in Escherichia coli, the modified ampR gene rapidly overproduced the AmpR protein as an insoluble aggregate. The AmpR protein could be solubilized with 1.32 M guanidine/HCl and remained soluble when dialyzed against 0.5 M NaCl. The solubility properties of AmpR were exploited to selectively precipitate and resolubilize the protein in a nearly homogenous state. AmpR was then purified by a single gel-filtration chromatography step which demonstrated that AmpR exists in solution as a monodisperse homodimeric protein. Several milligrams of purified AmpR could be obtained routinely from a 1-1 culture of induced bacteria. A DNA-binding assay buffer containing 300 mM potassium glutamate and 30% glycerol was found to stabilize AmpR and used to demonstrate sequence-specific DNA-binding. Additionally, purified AmpR binds a half-operator DNA with an inverted-repeat sequence which competes with binding by the wild-type operator. These findings are discussed in terms of the helix-turn-helix DNA-binding motif, whereby AmpR is proposed to interact with its wild-type operator as a dimer of dimers.

Isolation of TFC1, a gene encoding one of two DNA-binding subunits of yeast transcription factor tau (TFIIIC).

Transcription factor TFIIIC mediates tRNA and 5S RNA gene activation by binding to intragenic promoter elements. The factor from Saccharomyces cerevisiae, also called tau, is a large, multisubunit protein (550-650 kDa) containing two polypeptides that interact directly with DNA encoding tRNA (tDNA). We have obtained peptide sequences from the 95-kDa DNA-binding subunit (tau 95) and cloned the corresponding gene, called TFC1. The gene encodes a polypeptide of calculated Mr 73,500. However, when TFC1 was transcribed and translated in vitro, the gene product comigrated with tau 95 in SDS/polyacrylamide gels. A fusion protein expressed in bacteria was able to prevent the binding of anti-tau 95 antibodies to tau-tDNA complexes. The TFC1 gene is present in single copy on yeast chromosome II and is essential for growth. Spores containing a disrupted gene germinate but only proceed through a few cell divisions before ceasing to grow. The TFC1-encoded protein contains a potential helix-turn-helix structure and an acidic carboxyl-terminal domain, a feature characteristic of some DNA-binding proteins and transcriptional regulators.