Family Of Transcriptional Activators Research Articles

Hierarchical Control on Polyene Macrolide Biosynthesis: PimR Modulates Pimaricin Production via the PAS-LuxR Transcriptional Activator PimM

Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimR. This regulator combines an N-terminal domain corresponding to the Streptomyces antibiotic regulatory protein (SARP) family of transcriptional activators with a C-terminal half homologous to guanylate cyclases and large ATP-binding regulators of the LuxR family. The PimR SARP domain (PimRSARP) was expressed in Escherichia coli as a glutathione S-transferase (GST)–fused protein. Electrophoretic mobility shift assays showed that GST-PimRSARP binds a single target, the intergenic region between the regulatory genes pimR and pimMs in the pimaricin cluster. The PimRSARP-binding site was investigated by DNaseI protection studies, revealing that it contains three heptameric direct repeats adjusting to the consensus 5′-CGGCAAG-3′. Transcription start points of pimM and pimR promoters were identified by 5′-RACE, revealing that unlike other SARPs, PimRSARP does not interact with the -35 region of its target promoter. Quantitative transcriptional analysis of these regulatory genes on mutants on each of them has allowed the identification of the pimM promoter as the transcriptional target for PimR. Furthermore, the constitutive expression of pimM restored pimaricin production in a pimaricin-deficient strain carrying a deletion mutant of pimR. These results reveal that PimR exerts its positive effect on pimaricin production by controlling pimM expression level, a regulator whose gene product activates transcription from eight different promoters of pimaricin structural genes directly.

AxyR, an AraC Family Transcriptional Activator of the Xylanase 3 Gene, Requires Xylo-Oligosaccharides as a Cofactor for DNA Binding inPaenibacillussp. Strain W-61

The xylanolytic bacterium Paenibacillus sp. strain W-61 encodes three extracellular xylanase genes, xyn1, xyn3, and xyn5. In this study, we identified a transcriptional activator required for transcription of the xyn3 gene in strain W-61. The activator, AxyR, contained the highly homologous AraC-type DNA binding domain and required xylobiose, xylotriose, or xylotetraose as cofactor for binding to the xyn3 promoter region.

Abstract 1170: Anti-tumor effects of p53-pathway restoring compound Prodigiosin in colorectal cancer involve effects on apoptotic signaling, angiogenesis and cancer stem cells

Abstract Colorectal cancer (CRC) is the third leading cause of death due to cancer in USA. Restoration of wild-type (wt) p53 function alone has been shown to cause tumor regression in mice with minimal toxicity. p53-restoration compounds may provide a tumor-specific therapeutic regimen to overcome the resistance and toxicity associated with current CRC therapy. Utilizing a p53-responsive luciferase reporter-based screen we identified the fluorescent red bacterial pigment Prodigiosin as a potent p53 family transcriptional activator. Anti-tumoral properties of Prodigiosin have been reported for various types of cancer both in vitro and in vivo with minimal toxicity to normal tissues but the p53 mechanism has not been previously recognized. Prodigiosin has been shown to induce mitochondria-dependent apoptosis in cancer cells, but the role of the pro-apoptotic protein Bax in anti-tumor efficacy of Prodigiosin in CRC has not been clearly defined. Using fluorescence microscopy we determined that Prodigiosin localizes to the mitochondria in CRC cells. Results of trypan-blue dye exclusion tests reveal that HCT116 wild-type and Bax-null cells were equally sensitive to Prodigiosin-induced cell death. Thus, Bax played a minor role in Prodigiosin-induced cell death. The p53 family transcriptional activation by Prodigiosin is independent of the p53 status of the CRC cells. In vivo Prodigiosin induced p53 transcriptional activity and reduced tumor volume in subcutaneous xenografts of CRC cells. Prodigiosin induced apoptosis, inhibited cell proliferation and suppressed angiogenesis in vivo in CRC cells. Emerging evidence suggests that CRC tumors contain a rare population of CRC stem cells (CSCs) capable of self-renewal that are responsible for long-term sustenance of the tumor, local tumor recurrence and metastatic relapse. CSCs are therapy resistant since they are dormant and possess various efflux mechanisms. We hypothesize that anti-tumor effects of Prodigiosin involve targeting of the CSC population by p53 restoration in CSCs. With flow cytometric analysis for Prodigiosin fluorescence we determined that Prodigiosin is not effluxed from CRC cells. We have previously demonstrated that efflux of calcein AM is a marker for the cancer stem cell population. Here, we report that Prodigiosin is retained by the calcein low population (CloP) and induced apoptosis in CloP, as determined by fluorescence microscopy. Prodigiosin suppressed the ability of CRC cells to be serially passaged in vitro. Ongoing work involves determination of: mechanism of anti-angiogenic effect of Prodigiosin; effect of Prodigiosin on CSC frequency, self renewal and tumorigenicity; synergy of Prodigiosin with chemotherapeutic drugs for CRC in targeting CSCs. This work was supported by the NCI Developmental Therapeutics Program through NCI Contract N01-CN-43302. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1170. doi:1538-7445.AM2012-1170

Identification of the Regulator Gene Responsible for the Acetone-Responsive Expression of the Binuclear Iron Monooxygenase Gene Cluster in Mycobacteria

The mimABCD gene cluster encodes the binuclear iron monooxygenase that oxidizes propane and phenol in Mycobacterium smegmatis strain MC2 155 and Mycobacterium goodii strain 12523. Interestingly, expression of the mimABCD gene cluster is induced by acetone. In this study, we investigated the regulator gene responsible for this acetone-responsive expression. In the genome sequence of M. smegmatis strain MC2 155, the mimABCD gene cluster is preceded by a gene designated mimR, which is divergently transcribed. Sequence analysis revealed that MimR exhibits amino acid similarity with the NtrC family of transcriptional activators, including AcxR and AcoR, which are involved in acetone and acetoin metabolism, respectively. Unexpectedly, many homologs of the mimR gene were also found in the sequenced genomes of actinomycetes. A plasmid carrying a transcriptional fusion of the intergenic region between the mimR and mimA genes with a promoterless green fluorescent protein (GFP) gene was constructed and introduced into M. smegmatis strain MC2 155. Using a GFP reporter system, we confirmed by deletion and complementation analyses that the mimR gene product is the positive regulator of the mimABCD gene cluster expression that is responsive to acetone. M. goodii strain 12523 also utilized the same regulatory system as M. smegmatis strain MC2 155. Although transcriptional activators of the NtrC family generally control transcription using the σ(54) factor, a gene encoding the σ(54) factor was absent from the genome sequence of M. smegmatis strain MC2 155. These results suggest the presence of a novel regulatory system in actinomycetes, including mycobacteria.

The Serratia LuxR family regulator CarR39006 activates transcription independently of cognate quorum sensing signals

In Gram-negative bacteria, quorum sensing control of gene expression is mediated by transcription factors of the LuxR family, whose DNA-binding affinity is modulated by diffusible N-acyl homoserine lactone (AHL) signalling molecules. In Serratia sp. ATCC 39006 and the plant pathogen Erwinia carotovora ssp. carotovora (Ecc), the biosynthesis of the β-lactam antibiotic 1-carbapen-2-em-3-carboxylic acid (Car) is under quorum sensing control. This study has revealed that, uniquely, the LuxR family transcriptional activator CarR(39006) from Serratia 39006 has no detectable affinity for cognate AHL molecules. Furthermore, CarR(39006) was shown to be naturally competent to bind to its target promoter with high affinity, activate transcription and resist cellular proteolysis, and was unaffected by AHL signals. Experiments with chimeric proteins suggest that the C-terminal DNA-binding domain of CarR(39006) may be responsible for conferring AHL independence. In contrast, we show that the homologous CarR(Ecc) protein binds to its 3O-C6-HSL ligand with high affinity, and that the highly conserved Trp-44 residue is critical for this interaction. Unlike TraR from Agrobacterium tumefaciens, CarR(Ecc) is not directly protected from cellular proteolysis by AHL binding, but via AHL-induced DNA binding. At physiological protein concentrations, AHL binding induces CarR(Ecc) to bind to its target promoter with higher affinity and activate transcription.

The role of two SARP family transcriptional regulators in regulation of the auricin gene cluster in Streptomyces aureofaciens CCM 3239

Two regulators, Aur1P and Aur1R, have been previously found to control expression of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239 in a cascade mechanism. Here, we describe the characterization of two additional regulatory genes, aur1PR2 and aur1PR3, encoding homologues of the SARP family of transcriptional activators that were identified in the upstream part of the aur1 cluster. Expression of both genes is directed by a single promoter, aur1PR2p and aur1Pr3p, respectively, induced in late exponential phase. Disruption of aur1PR2 in S. aureofaciens CCM 3239 had no effect on auricin production. However, the disruption of aur1PR3 dramatically reduced auricin compared with its parental wild-type strain. Transcription from the aur1Ap promoter, directing expression of the first biosynthetic gene in the auricin gene cluster, was similarly decreased in the S. aureofaciens CCM 3239 aur1PR3 mutant. Transcription from the aur1PR3p promoter increased in the S. aureofaciens CCM 3239 aur1R mutant strain, and the TetR family negative regulator Aur1R was shown to specifically bind the aur1PR3p promoter. These results indicate a complex regulation of the auricin cluster by the additional SARP family transcriptional activator Aur1PR3.

CONSTANS and the evolutionary origin of photoperiodic timing of flowering

A network of promoting and inhibiting pathways that respond to environmental and internal signals controls the flowering transition. The outcome of this regulatory network establishes, for any particular plant, the correct time of the year to flower. The photoperiod pathway channels inputs from light, day length, and the circadian clock to promote the floral transition. CONSTANS (CO) is a central regulator of this pathway, triggering the production of the mobile florigen hormone FT (FLOWERING LOCUS T) that induces flower differentiation. Because plant reproductive fitness is directly related to its capacity to flower at a precise time, the photoperiod pathway is present in all known plant species. Recent findings have stretched the evolutionary span of this photophase signal to unicellular algae, which show unexpected conserved characteristics with modern plant photoperiodic responses. In this review, a comparative description of the photoperiodic systems in algae and plants will be presented and a general role for the CO family of transcriptional activators proposed.

Organization of the LEE1 operon regulatory region of enterohaemorrhagic Escherichia coli O157:H7 and activation by GrlA

Expression of the genes in the locus of enterocyte effacement (LEE) in enterohaemorrhagic Escherichia coli is primarily co-ordinated by expression of the LEE1 operon. GrlA is a LEE-encoded transcription regulator that has been proposed to be involved in the regulation of expression of the LEE1 operon. We describe a simple plasmid-based system to investigate the LEE1 operon regulatory region and to study GrlA-dependent effects. We report that GrlA can activate transcription initiation at the LEE1 P1 promoter by binding to a target located within the 18-base-pair spacer between the promoter -10 and -35 elements, which were defined by mutational analysis. Shortening this spacer to 17 base pairs increases P1 promoter activity and short-circuits GrlA-dependent activation. Hence, at the P1 promoter, the action of GrlA resembles that of many MerR family transcription activators at their target promoters.

ExsA Recruits RNA Polymerase to an Extended −10 Promoter by Contacting Region 4.2 of Sigma-70

ExsA is a member of the AraC family of transcriptional activators and is required for expression of the Pseudomonas aeruginosa type III secretion system (T3SS). ExsA-dependent promoters consist of two binding sites for monomeric ExsA located approximately 50 bp upstream of the transcription start sites. Binding to both sites is required for recruitment of sigma(70)-RNA polymerase (RNAP) to the promoter. ExsA-dependent promoters also contain putative -35 hexamers that closely match the sigma(70) consensus but are atypically spaced 21 or 22 bp from the -10 hexamer. Because several nucleotides located within the putative -35 region are required for ExsA binding, it is unclear whether the putative -35 region makes an additional contribution to transcription initiation. In the present study we demonstrate that the putative -35 hexamer is dispensable for ExsA-independent transcription from the P(exsC) promoter and that deletion of sigma(70) region 4.2, which contacts the -35 hexamer, has no effect on ExsA-independent transcription from P(exsC). Region 4.2 of sigma(70), however, is required for ExsA-dependent activation of the P(exsC) and P(exsD) promoters. Genetic data suggest that ExsA directly contacts region 4.2 of sigma(70), and several amino acids were found to contribute to the interaction. In vitro transcription assays demonstrate that an extended -10 element located in the P(exsC) promoter is important for overall promoter activity. Our collective data suggest a model in which ExsA compensates for the lack of a -35 hexamer by interacting with region 4.2 of sigma(70) to recruit RNAP to the promoter.

Three Dimensional Model for N-Terminal A Domain of DmpR (2-Dimethylphenol) Protein Based on Secondary Structure Prediction and Fold Recognition

DmpR (dimethylphenol regulatory protein) is a member of the NtrC family of transcriptional activators and controls the transcription of the dmp operons in response to aromatic effector compounds. Secondary structure and fold recognition prediction of N-terminal A domain of this protein (210 amino acid) was performed in Genesilico Metaserver and 3DJury. The consensus result from these servers suggested MJ_1460 as a template. Three dimensional structures were generated from the sequence structure alignments of the template and target protein with MODELLER. The results suggested that the N-terminal A domain of DmpR belongs to Muramoyl pentapeptide carboxypeptidase domain family. The binding interaction sites of the known effectors were predicted using protein-ligand docking. The proposed active site of N-terminal A domain of DmpR comprises of key residues such as Phe93, Glu127, Phe132, Ser160, Phe163, Met164, Arg166 and Pro189. The findings provide some direction to the experimental studies that aim to broaden the range of phenolic derivative which can be sensed by N-DmpR in order to improve the biodegradation potential.

The Gli2 transcriptional activator is a crucial effector for Ihh signaling in osteoblast development and cartilage vascularization

Indian hedgehog (Ihh) critically regulates multiple aspects of endochondral bone development. Although it is generally believed that all Ihh functions are mediated by the Gli family of transcription activators and repressors, formal genetic proof for this notion has not been provided. Moreover, the extent to which different Gli proteins contribute to Ihh functions is not fully understood. Previous work has shown that de-repression of the Gli3 repressor is the predominant mode through which Ihh controls chondrocyte proliferation and maturation, but that osteoblast differentiation and hypertrophic cartilage vascularization require additional mechanisms. To test the involvement of Gli2 activation in these processes, we have generated a mouse strain that expresses a constitutive Gli2 activator in a Cre-dependent manner, and have attempted to rescue the Ihh-null mouse with the Gli2 activator, either alone or in combination with Gli3 removal. Here, we report that the Gli2 activator alone is sufficient to induce vascularization of the hypertrophic cartilage in the absence of Ihh but requires simultaneous removal of Gli3 to restore osteoblast differentiation. These results therefore provide direct genetic evidence that Gli2 and Gli3 collectively mediate all major aspects of Ihh function during endochondral skeletal development.

Retinoblastoma Protein Plays Multiple Essential Roles in the Terminal Differentiation of Sertoli Cells

Retinoblastoma protein (RB) plays crucial roles in cell cycle control and cellular differentiation. Specifically, RB impairs the G(1) to S phase transition by acting as a repressor of the E2F family of transcriptional activators while also contributing towards terminal differentiation by modulating the activity of tissue-specific transcription factors. To examine the role of RB in Sertoli cells, the androgen-dependent somatic support cell of the testis, we created a Sertoli cell-specific conditional knockout of Rb. Initially, loss of RB has no gross effect on Sertoli cell function because the mice are fertile with normal testis weights at 6 wk of age. However, by 10-14 wk of age, mutant mice demonstrate severe Sertoli cell dysfunction and infertility. We show that mutant mature Sertoli cells continue cycling with defective regulation of multiple E2F1- and androgen-regulated genes and concurrent activation of apoptotic and p53-regulated genes. The most striking defects in mature Sertoli cell function are increased permeability of the blood-testis barrier, impaired tissue remodeling, and defective germ cell-Sertoli cell interactions. Our results demonstrate that RB is essential for proper terminal differentiation of Sertoli cells.

Activation of Gene Transcription by Heat Shock Protein 27 May Contribute to Its Neuronal Protection

Heat shock proteins are up-regulated as a physiological response to stressful stimuli and generally function as molecular chaperones for improperly folded protein substrates. The small heat shock protein HSP27 (or HSPB1) has multiple cytoplasmic roles. HSP27 also can translocate to the nucleus in response to stress, but the functional significance of this nuclear distribution has not been elucidated. We have previously implicated HSP27 as a genetic modifier of spinocerebellar ataxia 17 (SCA17), a neurological disease caused by a polyglutamine expansion in the TATA-binding protein (TBP). Altered expression of HSP27 is also found in cell models of other polyglutamine diseases, including Huntington disease as well as SCA3 and SCA7. Here, we show that Hsp27, unlike Hsp70, is not detected in mutant TBP aggregates in primary cerebellar granule neurons from transgenic SCA17 mice. Although HSP27 overexpression does not reduce the aggregation of cotransfected mutant TBP containing 105 glutamines, it potentiates activated transcription from both TATA-containing and TATA-lacking promoters. Neither HSP40 nor HSP70 elicits the same transcriptional effect. Moreover, HSP27 interacts with the transcription factor SP1, and coexpression of SP1 and nuclear localization signal-tagged HSP27 synergistically activates reporter constructs for the SP1-responsive neurotrophic receptor genes Ngfr(p75) and TRKA. Overexpression of nuclear localization signal-tagged HSP27 also rescues mutant TBP-mediated down-regulation of TrkA in a PC12 cell model of SCA17. These results indicate that nuclear HSP27 can modulate SP1-dependent transcriptional activity to promote neuronal protection.

Crystal structure of the hexamer of human heat shock factor binding protein 1

Heat shock response (HSR) is a ubiquitous cellular mechanism that copes with a variety of stresses. This response is mediated by a family of transcriptional activators, heat shock factors (HSFs), which are under tight regulation. HSF binding protein 1 (HSBP1) is a negative regulator of HSR and is reported to bind specifically with the active trimeric form of HSF1, thus inhibiting its activity. HSBP1 contains heptad-repeats in the primary sequence and was believed to stay in a trimer form in solution. We report the crystal structure of the trimerization domain of the M30I/L55P mutant of human HSBP1 at 1.8 A resolution. In this crystal form, the HSBP1 fragment of residues 6-53 forms a continuous, 11-turn long helix. The helix self-associates to form a parallel, symmetrical, triple coiled-coil helix bundle, which further assembles into a dimer of trimers in a head-to-head fashion. Solution study confirmed that the wild-type HSBP1 shares similar biophysical properties with the crystallized variant. Furthermore, we identified Ser31, which buried its polar side chain in the hydrophobic interior of the helix bundle, as a stability weak-spot. Substitution of this residue with Ile increases the melting temperature by 24 degrees C, implicating that this conserved serine residue is maintained at position 31 for functional purposes.

Positively regulated bacterial expression systems

SummaryRegulated promoters are useful tools for many aspects related to recombinant gene expression in bacteria, including for high‐level expression of heterologous proteins and for expression at physiological levels in metabolic engineering applications. In general, it is common to express the genes of interest from an inducible promoter controlled either by a positive regulator or by a repressor protein. In this review, we discuss established and potentially useful positively regulated bacterial promoter systems, with a particular emphasis on those that are controlled by the AraC‐XylS family of transcriptional activators. The systems function in a wide range of microorganisms, including enterobacteria, soil bacteria, lactic bacteria and streptomycetes. The available systems that have been applied to express heterologous genes are regulated either by sugars (l‐arabinose, l‐rhamnose, xylose and sucrose), substituted benzenes, cyclohexanone‐related compounds, ε‐caprolactam, propionate, thiostrepton, alkanes or peptides. It is of applied interest that some of the inducers require the presence of transport systems, some are more prone than others to become metabolized by the host and some have been applied mainly in one or a limited number of species. Based on bioinformatics analyses, the AraC‐XylS family of regulators contains a large number of different members (currently over 300), but only a small fraction of these, the XylS/Pm, AraC/PBAD, RhaR‐RhaS/rhaBAD, NitR/PnitA and ChnR/Pb regulator/promoter systems, have so far been explored for biotechnological applications.

Mitochondria and Hypoxia‐induced Gene Expression Mediated by Hypoxia‐inducible Factors

The influence of mitochondrial activity on gene expression programs, particularly those involved in neuroprotection and repair, is likely to play an important role in the pathophysiology of neurodegenerative diseases. One such gene expression program is activated by the cellular pathway that senses a decrease in optimal oxygen levels and leads to activation of a family of transcriptional activators called hypoxia-inducible factors (HIFs). HIFs are members of the bHLH-PAS family of transcription factors and are heterodimers composed of HIF-alpha and HIF-beta (also known as aryl hydrocarbon receptor nuclear translocator) subunits that bind to canonical DNA sequences (hypoxia-regulated elements) in the promoters or enhancers of target genes. HIFs activate the expression of more than a hundred genes encoding proteins that regulate cell metabolism, survival, angiogenesis, vascular tone, hematopoiesis, and other functions. There is considerable evidence showing a bidirectional crosstalk between mitochondrial signals and HIF activity. For instance, mitochondrial reactive oxygen species and metabolic substrates from the tricarboxylic acid cycle are implicated in the regulation of the HIF pathway. Conversely, HIF activity leads to the expression of target genes that influence mitochondrial function. In this chapter we will review the complex interactions between mitochondria and the HIF pathway and we will discuss the relevance of this interaction for metabolic adaptation to hypoxia.

Relaxed Selection on the CBF/DREB1 Regulatory Genes and Reduced Freezing Tolerance in the Southern Range of Arabidopsis thaliana

Elucidating the molecular basis of adaptive phenotypic variation represents a central aim in evolutionary biology. Traits exhibiting patterns of clinal variation represent excellent models for studies of molecular adaptation, especially when variation in phenotype can be linked to organismal fitness in different environments. Natural accessions of the model plant species Arabidopsis thaliana exhibit clinal variation in freezing tolerance that follows a gradient of temperature variability across the species' native range (Zhen Y, Ungerer MC. 2008. Clinal variation in freezing tolerance among natural accessions of A. thaliana. New Phytol. 177:419-427). Here, we report that this pattern of variation is attributable, at least in part, to relaxed purifying selection on members of a small family of transcriptional activators (the CBF/DREB1s) in the species' southern range. These regulatory genes play a critical role in the ability of A. thaliana plants to undergo cold acclimation and thereby achieve maximum freezing tolerance. Relative to accessions from northern regions, accessions of A. thaliana from the southern part of their geographic range exhibit levels of nonsynonymous nucleotide polymorphism that are approximately 2.8-fold higher across this small gene subfamily. Relaxed selection on the CBF/DREB1s in southern accessions also has resulted in multiple mutations in regulatory regions resulting in abrogated expression of particular subfamily members in particular accessions. These coding-region and regulatory mutations compromise the ability of these genes to act as efficient transcriptional activators during the cold acclimation process, as determined by reductions in rates of induction and maximum levels of expression in the downstream genes they regulate. This study highlights the potential role of regulatory genes in underlying adaptive phenotypic variation in nature.

Identification of transcriptional activators for thienamycin and cephamycin C biosynthetic genes within the thienamycin gene cluster from Streptomyces cattleya

Two regulatory genes, thnI and thnU, were identified in the thienamycin (thn) gene cluster from Streptomyces cattleya. ThnI resembles LysR-type transcriptional activators and ThnU belongs to the SARP family of transcriptional activators. Their functional role was established after independent inactivation by gene replacement together with transcriptional analysis involving reverse transcription polymerase chain reaction (RT-PCR). Deletion of thnI abolished thienamycin production showing its involvement in thienamycin biosynthesis. Gene expression analysis applied to the thn gene cluster demonstrated that ThnI is a transcriptional activator essential for thienamycin biosynthesis that regulates the expression of nine genes involved in thienamycin assembly and export (thnH, thnJ, thnK, thnL, thnM, thnN, thnO, thnP and thnQ). Unexpectedly, the thnU disrupted mutant was not affected in thienamycin production but turned out to be essential for cephamycin C biosynthesis. Transcript analysis applied to early and late structural genes for cephamycin C biosynthesis (pcbAB and cmcI), revealed that ThnU is the transcriptional activator of these cephamycin C genes although they are not physically linked to the thn cluster. In addition, it was shown that deletion of thnI has an upregulatory effect on pcbAB and cmcI transcription consistent with a significant increase in cephamycin C biosynthesis in this mutant.

Differences in the mechanism of the allosteric l‐rhamnose responses of the AraC/XylS family transcription activators RhaS and RhaR

Proteins in the largest subset of AraC/XylS family transcription activators, including RhaS and RhaR, have C-terminal domains (CTDs) that mediate DNA-binding and transcription activation, and N-terminal domains (NTDs) that mediate dimerization and effector binding. The mechanism of the allosteric effector response in this family has been identified only for AraC. Here, we investigated the mechanism by which RhaS and RhaR respond to their effector, l-rhamnose. Unlike AraC, N-terminal truncations suggested that RhaS and RhaR do not use an N-terminal arm to inhibit activity in the absence of effector. We used random mutagenesis to isolate RhaS and RhaR variants with enhanced activation in the absence of l-rhamnose. NTD substitutions largely clustered around the predicted l-rhamnose-binding pockets, suggesting that they mimic the structural outcome of effector binding to the wild-type proteins. RhaS-CTD substitutions clustered in the first HTH motif, and suggested that l-rhamnose induces improved DNA binding. In contrast, RhaR-CTD substitutions clustered at a single residue in the second HTH motif, at a position consistent with improved RNAP contacts. We propose separate allosteric mechanisms for the two proteins: Without l-rhamnose, RhaS does not effectively bind DNA while RhaR does not effectively contact RNAP. Upon l-rhamnose binding, both proteins undergo structural changes that enable transcription activation.

Analysis of the Campylobacter jejuni FlgR Response Regulator Suggests Integration of Diverse Mechanisms To Activate an NtrC-Like Protein

Flagellar motility in Campylobacter jejuni mediates optimal interactions with human or animal hosts. Sigma(54) and the FlgSR two-component system are necessary for the expression of many C. jejuni flagellar genes. The FlgR response regulator is homologous to the NtrC family of transcriptional activators. These regulators usually contain an N-terminal receiver domain, a central domain that interacts with sigma(54) and hydrolyzes ATP, and a DNA-binding C-terminal domain. Most often, phosphorylation of the receiver domain influences its inherent ability to either positively or negatively control the activity of the regulator. In this study, we performed genetic and biochemical analyses to understand how FlgR activity is controlled to culminate in the expression of sigma(54)-dependent flagellar genes. Our data suggest that the FlgR receiver domain has the capacity for both positive and negative regulation in controlling the activation of the protein. Analysis of the C-terminal domain of FlgR revealed that it lacks a DNA-binding motif and is not required for sigma(54)-dependent flagellar gene expression. Further analysis of FlgR lacking the C-terminal domain indicates that this protein is partially functional in the absence of the cognate sensor kinase, FlgS, but its activity is still dependent on the phosphorylated residue in the receiver domain, D51. We hypothesize that the C-terminal domain may not function to bind DNA but may ensure the specificity of the phosphorylation of FlgR by FlgS. Our results demonstrate that FlgR activation mechanisms are unusual among characterized NtrC-like proteins and emphasize that various means are utilized by the NtrC family of proteins to control the transcription of target genes.