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Methanol is a promising substrate for sustainable biomanufacturing, and Pichia pastoris has become a commonly used yeast for methanol utilization due to its powerful methanol metabolic pathways and methanol inducible promoter. Previous reconstruction of gene circuits highly improved transcriptional activity, but excessive expression of chimeric transactivator damaged cell growth on methanol. Here we employed transcriptome analysis to investigate the effects of chimeric transactivator overexpression on cellular metabolism and regulatory networks. The results showed that strong expression of chimeric transactivator unexpectedly downregulated methanol metabolism, especially the alcohol oxidase 1 (AOX1), but without remarkable changes in expression of transcriptional factors. Meanwhile, the synthesis of peroxisomes also varied with chimeric transactivator expression. In addition, the enrichment analysis of differentially expressed genes revealed their impact on cellular metabolism. The gene expression patterns caused by different expression levels of chimeric transactivators have also been clarified. This work provides useful information to understand the transcriptional regulation of the AOX1 promoter and methanol signaling. It revealed the importance of balancing transcription factor expression for the host improvement.

BACKGROUND: Most of the formulas for the clinical treatment of premature ovarian insufficiency have evolved from the basic formula of Liuwei Dihuang Pills, and have achieved good therapeutic efficacy. Currently, most of the experimental studies on Liuwei Dihuang Pills focus on morphological observations and physiological and biochemical detection of in vivo animal models, while fewer studies on molecular mechanisms have been reported. OBJECTIVE: To explore the molecular mechanism of Liuwei Dihuang Pills in the treatment of premature ovarian insufficiency based on the receptor gamma coactivator-1 alpha/mitochondrial transcription factor A/reactive oxygen species pathway. METHODS: Premature ovarian insufficiency model was established in mice by intraperitoneal injection of cyclophosphamide 120 mg/kg combined with busulfan 12 mg/kg, and then Liuwei Dihuang Pill suspension was used to intervene in premature ovarian insufficiency mice. After 12 weeks of intervention, the levels of follicle-stimulating hormone, luteinizing hormone, estradiol, anti-Mullerian hormone, 8-hydroxydeoxyguanosine, total antioxidant capacity and reactive oxygen species in serum of mice were detected by ELISA method. The morphological changes in mouse ovaries were observed by hematoxylin-eosin staining. The ultrastructure of mouse follicular granulosa cells and the apoptosis of granulosa cell mitochondria were observed by transmission electron microscopy. The expression levels of receptor gamma coactivator-1 alpha and mitochondrial transcription factor A in mouse ovarian granulosa cells were detected by immunohistochemistry. RESULTS AND CONCLUSION: Compared with the model group, serum levels of follicle-stimulating hormone, luteinizing hormone, reactive oxygen species, and 8-hydroxydeoxyguanosine were decreased in the experimental group (P < 0.05), and the levels of estradiol, anti-Mullerian hormone, and total antioxidant capacity were increased (P < 0.05). Hematoxylin-eosin staining showed that in the model group, there were more atretic follicles and corpus luteum forms, some secondary follicles, and interstitial fibrosis and hyperplasia; in the experimental group, a large number of atretic follicles, few corpus luteum forms, primordial follicles were observed at the edges but there were few secondary follicles and no mature follicles. Transmission electron microscopy showed that the organelles in ovarian granulosa cells of mice in the experimental groups were relatively intact. Immunohistochemical results showed that compared with the model group, the expression level of receptor gamma coactivator-1 alpha in the ovarian tissue of mice increased slightly in the experimental group at the 4th week, and there was no significant change at the 8th and 12th weeks. The expression level of mitochondrial transcription factor A in the ovarian tissues of mice in the experimental group was transiently increased at the 4th week, and then slightly decreased, which were all significantly different from those of the model group. To conclude, Liuwei Dihuang Pills inhibit ovarian granulosa cell apoptosis in mice with premature ovarian insufficiency to a certain extent through the receptor gamma coactivator-1 alpha/mitochondrial transcription factor A/reactive oxygen species signaling pathway, thereby improving the endocrine function of the ovary, enhancing the antioxidant capacity, and attenuating the degree of oxidative stress damage. [ABSTRACT FROM AUTHOR]

Abstract Background The filamentous fungus Trichoderma reesei is extensively used for the industrial-scale cellulase production. It has been well known that the transcription factor Xyr1 plays an important role in the regulatory network controlling cellulase gene expression. However, the role of Xyr1 in the regulation of cellulase expression has not been comprehensively elucidated, which hinders further improvement of lignocellulolytic enzyme production. Results Here, the expression dosage of xyr1 was tailored in T. reesei by differentially overexpressing the xyr1 gene under the control of three strong promoters (Pegl2, Pcbh1, and Pcdna1), and the transcript abundance of xyr1 was elevated 5.8-, 12.6-, and 47.2-fold, respectively. We found expression of cellulase genes was significantly increased in the Pegl2-driven xyr1 overexpression strain QE2X, whereas relatively low in the Pcbh1- and Pcdna1-driven overexpression strains. We also found that the Pegl2-driven overexpression of xyr1 caused a more significant opening of chromatin in the core promoter region of the prominent cellulase genes. Furthermore, the cellulase activity showed a 3.2-fold increase in the strain QE2X, while insignificant improvement in the Pcbh1- and Pcdna1-driven strains. Finally, the saccharification efficiency toward acid-pretreated corncob residues containing high-content lignin by the crude enzyme from QE2X was increased by 57.2% compared to that from the parental strain. Moreover, LC–MS/MS and RT-qPCR analysis revealed that expression of accessory proteins (Cip1, Cip2, Swo1, and LPMOs) was greatly improved in QE2X, which partly explained the promoting effect of the Pegl2-driven overexpression on enzymatic hydrolysis of lignocellulose biomass. Conclusions Our results underpin that the precise tailoring expression of xyr1 is essential for highly efficient cellulase synthesis, which provide new insights into the role of Xyr1 in regulating cellulase expression in T. reesei. Moreover, these results also provides a prospective strategy for strain improvement to enhance the lignocellulolytic enzyme production for use in biorefinery applications.

Background: The filamentous fungus Trichoderma reesei is extensively used for the industrial-scale cellulase production. It has been well known that the transcription factor Xyr1 plays an important role in the regulatory network controlling cellulase gene expression. However, the role of Xyr1 in the regulation of cellulase expression has not been comprehensively elucidated, which hinders further improvement of lignocellulolytic enzyme production. Results: Here, the expression dosage of xyr1 was tailored in T. reesei by differentially overexpressing the xyr1 gene under the control of three strong promoters (Pegl2, Pcbh1, and Pcdna1), and the transcript abundance of xyr1 was elevated 5.8-, 12.6-, and 47.2-fold, respectively. We found expression of cellulase genes was significantly increased in the Pegl2-driven xyr1 overexpression strain QE2X, whereas relatively low in the Pcbh1- and Pcdna1-driven overexpression strains. We also found that the Pegl2-driven overexpression of xyr1 caused a more significant opening of chromatin in the core promoter region of the prominent cellulase genes. Furthermore, the cellulase activity showed a 3.2-fold increase in the strain QE2X, while insignificant improvement in the Pcbh1- and Pcdna1-driven strains. Finally, the saccharification efficiency toward acid-pretreated corncob residues containing high-content lignin by the crude enzyme from QE2X was increased by 57.2% compared to that from the parental strain. Moreover, LC–MS/MS and RT-qPCR analysis revealed that expression of accessory proteins (Cip1, Cip2, Swo1, and LPMOs) was greatly improved in QE2X, which partly explained the promoting effect of the Pegl2-driven overexpression on enzymatic hydrolysis of lignocellulose biomass. Conclusions: Our results underpin that the precise tailoring expression of xyr1 is essential for highly efficient cellulase synthesis, which provide new insights into the role of Xyr1 in regulating cellulase expression in T. reesei. Moreover, these results also provides a prospective strategy for strain improvement to enhance the lignocellulolytic enzyme production for use in biorefinery applications. [ABSTRACT FROM AUTHOR]

The Keap1-Nrf2 complex is a central regulator that mediates transcriptional responses to xenobiotic stimuli and is highly related with multiple human diseases. The molecular mechanisms and biological functions of Keap1 and Nrf2 are not fully understood. The Drosophila Keap1 homolog (dKeap1) is conserved with mammalian Keap1 except that dKeap1 contains a 156 aa C-terminal tail (CTD). A dKeap1 truncation with the CTD removed (dKeap1-ΔCTD) shows abolished nuclear localization and chromatin-binding. Expression of dKeap1-ΔCTD in the dKeap1 null background significantly rescues this mutant to the adult stage, but the files showed partial lethality, sterility and defects in adipose tissue. In the rescued flies, expression levels of ecdysone-response genes, ecdysone-synthetic genes and adipogenesis genes were down-regulated in specific tissues, indicating that the chromatin-binding of dKeap1 mediates the activation of these developmental genes. At the same time, dKeap1-ΔCTD can still suppress the basal expression of detoxifying genes and mediate the activation of these genes in response to xenobiotic stimuli, suggesting that the chromatin-binding of dKeap1 is not required for the regulation of detoxifying genes. These results support a model in which dKeap1 on one hand functions as an inhibitor for the Nrf2-mediated transcription in the xenobiotic response pathway and on the other hand functions as a chromatin-binding transcription activator in the developmental pathway. Our study reveals a novel mechanism whereby Keap1-Nrf2 xenobiotic response signaling regulates development using a mechanism independent of redox signaling. [Display omitted] • Drosophila Keap1 binds to chromatin using the C-terminal domain. • dKeap1 that loses chromatin-binding functions normally in the xenobiotic response pathway. • Chromatin-bound dKeap1 facilitates the activation of developmental genes. • dKeap1 and CncC activates ecdysone-synthetic genes during oogenesis. • dKeap1 regulates adipogenesis genes in the fat tissue. [ABSTRACT FROM AUTHOR]

Methanol is a promising substrate for sustainable biomanufacturing, and Pichia pastoris has become a commonly used yeast for methanol utilization due to its powerful methanol metabolic pathways and methanol inducible promoter. Previous reconstruction of gene circuits highly improved transcriptional activity, but excessive expression of chimeric transactivator damaged cell growth on methanol. Here we employed transcriptome analysis to investigate the effects of chimeric transactivator overexpression on cellular metabolism and regulatory networks. The results showed that strong expression of chimeric transactivator unexpectedly downregulated methanol metabolism, especially the alcohol oxidase 1 ( AOX1 ), but without remarkable changes in expression of transcriptional factors. Meanwhile, the synthesis of peroxisomes also varied with chimeric transactivator expression. In addition, the enrichment analysis of differentially expressed genes revealed their impact on cellular metabolism. The gene expression patterns caused by different expression levels of chimeric transactivators have also been clarified. This work provides useful information to understand the transcriptional regulation of the AOX1 promoter and methanol signaling. It revealed the importance of balancing transcription factor expression for the host improvement., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Temozolomide (TMZ)-based chemotherapy is a standard strategy for gliomas, although chemoresistance remains a major therapeutic challenge. The chemical mechanism by which TMZ induces cell death is DNA methylation, leading to double-stranded breaks (DSBs) and thus to apoptosis. However, TMZ-induced N6-meG sites are efficiently repaired and mediated by the DNA repair protein O6-methylguanine-DNA methyl-transferase (MGMT), leading to TMZ resistance. KLF15, a member of the Kruppel-like factors family, mainly functions as transcription factor and potential suppressor gene by inhibiting proliferation, migration, and inducing apoptosis. However, the roles and regulatory mechanisms of KLF15 in glioma tumorigenesis and chemoresistance are poorly understood. In this study, KLF15 expression was upregulated in glioma tissues and cell lines upon TMZ treatment. Knockdown of KLF15 amplified TMZ-induced repression of cell proliferation, while KLF15 overexpression reversed this process. Mechanistically, KLF15 functioned as a transcriptional activator of MGMT. Moreover, KLF15 knockdown sensitized tumors to TMZ treatment in vivo. Taken together, these results suggested that KLF15 up-regulated MGMT through direct binding to the promoter of MGMT, which plays an important role in glioma resistance to TMZ, and which may be a potential target for cancer diagnosis and treatment.

Hypoxia-inducible factor (HIF), an αβ dimer, is the master regulator of oxygen homeostasis with hundreds of hypoxia-inducible target genes. Three HIF isoforms differing in the oxygen-sensitive α subunit exist in vertebrates. While HIF-1 and HIF-2 are known transcription activators, HIF-3 has been considered a negative regulator of the hypoxia response pathway. However, the human HIF3A mRNA is subject to complex alternative splicing. It was recently shown that the long HIF-3α variants can form αβ dimers that possess transactivation capacity. Here, we show that overexpression of the long HIF-3α2 variant induces the expression of a subset of genes, including the erythropoietin (EPO) gene, while simultaneous downregulation of all HIF-3α variants by siRNA targeting a shared HIF3A region leads to downregulation of EPO and additional genes. EPO mRNA and protein levels correlated with HIF3A silencing and HIF-3α2 overexpression. Chromatin immunoprecipitation analyses showed that HIF-3α2 binding associated with canonical hypoxia response elements in the promoter regions of EPO. Luciferase reporter assays showed that the identified HIF-3α2 chromatin-binding regions were sufficient to promote transcription by all three HIF-α isoforms. Based on these data, HIF-3α2 is a transcription activator that directly regulates EPO expression. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To investigate the effect of sevoflurane on hepatic ischemia-reperfusion injury in rats via janus kinase 2/signal transducer and activator of transcription 3 (JAK2-STAT3) pathway. MATERIALS AND METHODS: Forty healthy male Sprague-Dawley (SD) rats were randomly divided into sham group (n=10), model group (HIRI group, n=10), sevoflurane intervention group (SF group, n=10), and sevoflurane combined with AG490 intervention group (AG490 group, n=10). Liver and serum samples were collected after reperfusion for 6 h. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), interleukin (IL) -1β, IL-6 and tumor necrosis factor (TNF) -α were detected by enzyme-linked immunosorbent assay (ELISA). JAK2, STAT3, p-JAK2 and p-STAT3 were detected by Western-blot. The expression level of cobalt quenching technique was used to detect the permeability of mitochondrial membrane permeability transition pore (mPTP). RESULTS: The levels of ALT, AST, AKP, IL- 1β, IL-6 and TNF-α in HIRI group were higher than those in sham group (p < 0.05), those in SF group were lower than those in HIRI group (p < 0.05), while those in AG490 group were higher than those in SF group (p < 0.05). The levels of JAK2 protein, pJAK2 protein, STAT3 protein and P STAT3 protein in HIRI group were lower than those in sham group (p < 0.05); the levels of each protein in SF group were higher than those in HIRI group (p < 0.05), while those in AG490 group were lower than those in SF group (p < 0.05). The open degree of HIRI group was higher than that of sham group (p < 0.05); SF group was lower than that of HIRI group, and AG490 group was higher than that of SF group (p < 0.05). CONCLUSIONS: Sevoflurane can significantly improve HIRI and reduce hepatic immune inflammation in rats. The mechanism may be related to activating JAK2-STAT3 pathway and inhibiting the overopening of mPTP. [ABSTRACT FROM AUTHOR]

Certain plant-associated Proteobacteria sense their host environment by detecting an unknown plant signal recognized by a member of a LuxR subfamily of transcription factors. This interkingdom communication is important for both mutualistic and pathogenic interactions. The Populus root endophyte Pseudomonas sp. GM79 possesses such a regulator, named PipR. In a previous study we reported that PipR activates an adjacent gene (pipA) coding for a proline iminopeptidase in response to Populus leaf macerates and peptides and that this activation is dependent on a putative ABC-type transporter [Schaefer AL, et al. (2016) mBio 7:e01101-16]. In this study we identify a chemical derived from ethanolamine that induces PipR activity at picomolar concentrations, and we present evidence that this is the active inducer present in plant leaf macerates. First, a screen of more than 750 compounds indicated ethanolamine was a potent inducer for the PipR-sensing system; however, ethanolamine failed to bind to the periplasmic-binding protein (PBP) required for the signal response. This led us to discover that a specific ethanolamine derivative, N-(2-hydroxyethyl)-2-(2-hydroxyethylamino) acetamide (HEHEAA), binds to the PBP and serves as a potent PipR-dependent inducer. We also show that a compound, which coelutes with HEHEAA in HPLC and induces pipA gene expression in a PipR-dependent manner, can be found in Populus leaf macerates. This work sheds light on how plant-associated bacteria can sense their environment and on the nature of inducers for a family of plant-responsive LuxR-like transcription factors found in plant-associated bacteria. [ABSTRACT FROM AUTHOR]

Eukaryotic transcription activation domains (ADs) are intrinsically disordered polypeptides that typically interact with coactivator complexes, leading to stimulation of transcription initiation, elongation, and chromatin modifications. Here we examined the properties of two strong and conserved yeast ADs: Met4 and Ino2. Both factors have tandem ADs that were identified by conserved sequence and functional studies. While the AD function of both factors depended on hydrophobic residues, Ino2 further required key conserved acidic and polar residues for optimal function. Binding studies showed that the ADs bound multiple Med15 activatorbinding domains (ABDs) with similar orders of micromolar affinity and similar but distinct thermodynamic properties. Protein cross-linking data show that no unique complex was formed upon Met4-Med15 binding. Rather, we observed heterogeneous AD-ABD contacts with nearly every possible AD-ABD combination. Many of these properties are similar to those observed with yeast activator Gcn4, which forms a large heterogeneous, dynamic, and fuzzy complex with Med15. We suggest that this molecular behavior is common among eukaryotic activators. [ABSTRACT FROM AUTHOR]

Delivery of chemotherapeutic drugs using nanocarriers is emerging as a promising approach for the treatment of cancer. The aim of this research was to develop a dual targeted d -α-tocopheryl succinate (TOS) functionalized nanostructured lipid carriers (NLCs), using etoposide (ETP) as a model drug to prove their in vitro and in vivo anti-tumor effects. Novel ETP loaded NLCs were constructed (ETP-NLCs). Hyaluronic acid (HA) and cell-penetrating peptide transcription activator (TAT) was applied for the surface decoration of ETP NLCs (HATOS/TATTOS-ETP-NLCs). The antitumor efficiency of HATOS/TATTOS-ETP-NLCs was evaluated in tumor bearing animal models. HATOS/TATTOS-ETP-NLCs displayed significantly higher transfection efficiency and better antitumor ability than undecorated ETP-NLCs in lymphoma cells bearing mice model. The newly constructed NLCs could successfully load drug and gene; and TAT could function as excellent targeting ligands to improve the cell targeting ability of the gene loaded nanocarriers. The resulting dual ligands decorated vectors could be a promising targeted gene delivery system for the lymphoma treatment. [ABSTRACT FROM AUTHOR]

Members of the Drosophila behavior/human splicing protein family, including splicing factor proline/glutamine rich ( SFPQ), non- POU domain-containing octamer-binding protein ( NONO), and paraspeckle protein component 1 ( PSPC1), are abundantly expressed in testicular Sertoli cells ( SCs), but their roles remain obscure. Here, we show that treatment with mono-(2-ethylhexyl) phthalate ( MEHP), a well-known SC toxicant, selectively stimulates the expression levels of NONO and PSPC1. Simultaneous inhibition of NONO and PSPC1 expression in SCs enhances MEHP-induced oxidative stress and potentiates SC death. Mechanistically, NONO and PSPC1 transcriptionally activate aldehyde dehydrogenase 1 ( Aldh1a1), by synergistically binding to the distinct CCGGAGTC sequence in the Aldh1a1 promoter. Together, the NONO/ PSPC1- ALDH1A1 cascade may serve as an indispensable defense mechanism against MEHP insult in SCs. [ABSTRACT FROM AUTHOR]

Regulation of transcription is vitally important for maintaining normal cellular homeostasis and is also the basis for cellular differentiation, morphogenesis and the adaptability of any organism. Transcription activators, which orchestrate time and locus-specific assembly of complex transcription machinery, act as key players in these processes. One way in which these activators are controlled is by the covalent attachment of the conserved protein, ubiquitin (Ub), which can serve as either a proteolytic or non-proteolytic signal. For a subset of the activators, polyubiquitination-dependent degradation of the activator controls its abundance. In these cases transcription activation can require protein synthesis as well as internal or external stimulus. In contrast, other activators have been reported to undergo mono- or oligoubiquitination that does not lead to protein degradation. The mechanisms by which monoubiquitination of transcription activators affect their activities have been poorly understood. In a recent study, we demonstrated that monoubiquitination of some transcription activators can inhibit transcription by recruiting the AAA+ ATPase Cdc48 (also known in metazoan organisms as p97 or valosin-contain protein, VCP), which then extracts the ubiquitinated activator from DNA.

Background: Immune dysregulation plays an important role in the pathogenesis of rheumatoid arthritis (RA). The CD4+CD25 high FoxP3+ subset of regulatory T cells plays an essential role in preventing autoimmunity and maintaining immune homeostasis. Negative regulation of JAK/STAT signaling is controlled by Suppressor of Cytokine Signaling (SOCs3) proteins. SOCs is produced at lower levels in RA. Our aim was to evaluate the expressional dysregulation of SOCs3 and FoxP3 genes in RA patients in relation to disease activity., Methods: We have recruited 90 patients with RA and 60 healthy controls in case control study. Whole blood samples were collected from RA patients and healthy subjects. The measurement of FoxP3 and SOCs3 gene expression was performed by real-time PCR (qPCR)., Results: Patients with RA had significantly decreased expression levels of FoxP3 and SOCs3 genes in comparison with controls (P<0.001), in addition to the insignificance correlation of both genes with disease activity in RA patients., Conclusion: FoxP3 and SOCs3 genes showed significant defects in rheumatoid arthritis patients with no significant difference in disease activity., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Vibrio parahaemolyticus (Vp) is a marine halophilic bacterium that is commonly associated with oysters and shrimp. Human consumption of contaminated shellfish can result in Vp mediated gastroenteritis and severe diarrheal disease. Vp encodes two type 3 secretion systems (T3SS-1 and T3SS2) that have been functionally implicated in cytotoxicity and enterotoxicity respectively. In this study, we profiled protein secretion and temporal promoter activities associated with exsA and exsB gene expression. exsA is an AraC-like transcriptional activator that is critical for activating multiple operons that encode T3SS-1 genes, whereas exsB is thought to encode an outer membrane pilotin component for T3SS-1. The exsBA genetic locus has two predicted promoter elements. The predicted exsB and exsA promoters were individually cloned upstream of luxCDABE genes in reporter plasmid constructs allowing for in situ, real-time quantitative light emission measurements under many growth conditions. Low calcium growth conditions supported maximal exsB and exsA promoter activation. exsB promoter activity exhibited high basal activity and resulted in an exsBA co-transcript. Furthermore, a separate proximal exsA promoter showed initial low basal activity yet eventually exceeded that of exsB and reached maximal levels after 2.5 h corresponding to an entry into early log phase. exsA promoter activity was significantly higher at 30°C than 37°C, which also coincided with increased secretion levels of specific T3SS-1 effector proteins. Lastly, bioinformatic analyses identified a putative expanded ExsA binding motif for multiple transcriptional operons. These findings suggest a two wave model of Vp T3SS-I induction that integrates two distinct promoter elements and environmental signals into a complex ExsA activation framework. [ABSTRACT FROM AUTHOR]

The pH-responsive one-component signaling system CadC in Escherichia coli belongs to the family of ToxR-like proteins, whose members share a conserved modular structure, with an N-terminal cytoplasmic winged helix–turn–helix DNA-binding domain being followed by a single transmembrane helix and a C-terminal periplasmic pH-sensing domain. In E. coli CadC, a cytoplasmic linker comprising approximately 50 amino acids is essential for transmission of the signal from the sensor to the DNA-binding domain. However, the mechanism of transduction is poorly understood. Using NMR spectroscopy, we demonstrate here that the linker region is intrinsically disordered in solution. Furthermore, mutational analyses showed that it tolerates a range of amino acid substitutions (altering polarity, rigidity and α-helix-forming propensity), is robust to extension but is sensitive to truncation. Indeed, truncations either reversed the expression profile of the target operon cadBA or decoupled expression from external pH altogether. CadC dimerizes via its periplasmic domain, but light-scattering analysis provided no evidence for dimerization of the isolated DNA-binding domain, with or without the linker region. However, bacterial two-hybrid analysis revealed that CadC forms stable dimers in a stimulus- and linker-dependent manner, interacting only at pH < 6.8. Strikingly, a variant with inversed cadBA expression profile, which lacks most of the linker, dimerizes preferentially at higher pH. Thus, we propose that the disordered CadC linker is required for transducing the pH-dependent response of the periplasmic sensor into a structural rearrangement that facilitates dimerization of the cytoplasmic CadC DNA-binding domain. [ABSTRACT FROM AUTHOR]

The hexitol D-mannitol is transported by many bacteria via a phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). In most Firmicutes, the transcription activator MtlR controls the expression of the genes encoding the D-mannitol-specific PTS components and D-mannitol-1-P dehydrogenase. MtlR contains an N-terminal helix-turn-helix motif followed by an Mga-like domain, two PTS regulation domains (PRDs), an EIIBGat- and an EIIAMtl-like domain. The four regulatory domains are the target of phosphorylation by PTS components. Despite strong sequence conservation, the mechanisms controlling the activity of MtlR from Lactobacillus casei, Bacillus subtilis and Geobacillus stearothermophilus are quite different. Owing to the presence of a tyrosine in place of the second conserved histidine (His) in PRD2, L. casei MtlR is not phosphorylated by Enzyme I (EI) and HPr. When the corresponding His in PRD2 of MtlR from B. subtilis and G. stearothermophilus was replaced with alanine, the transcription regulator was no longer phosphorylated and remained inactive. Surprisingly, L. casei MtlR functions without phosphorylation in PRD2 because in a ptsI (EI) mutant MtlR is constitutively active. EI inactivation prevents not only phosphorylation of HPr, but also of the PTSMtl components, which inactivate MtlR by phosphorylating its EIIBGat- or EIIAMtl-like domain. This explains the constitutive phenotype of the ptsI mutant. The absence of EIIBMtl-mediated phosphorylation leads to induction of the L. caseimtl operon. This mechanism resembles mtlARFD induction in G. stearothermophilus, but differs from EIIAMtl-mediated induction in B. subtilis. In contrast to B. subtilis MtlR, L. casei MtlR activation does not require sequestration to the membrane via the unphosphorylated EIIBMtl domain. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Plant immune responses against biotrophic pathogens are regulated by the signaling hormone salicylic acid (SA). SA establishes immunity by regulating a variety of cellular processes, including programmed cell death (PCD) to isolate and kill invading pathogens, and development of systemic acquired resistance (SAR) which provides long-lasting, broad-spectrum resistance throughout the plant. Central to these processes is post-translational modification of SA-regulated signaling proteins by ubiquitination, i.e., the covalent addition of small ubiquitin proteins. Emerging evidence indicates SA-induced protein ubiquitination is largely orchestrated by Cullin-RING ligases (CRLs), which recruit specific substrates for ubiquitination using interchangeable adaptors. Ligation of ubiquitin chains interlinked at lysine 48 leads to substrate degradation by the 26S proteasome. Here we discuss how CRL-mediated degradation of both nucleotide-binding/leucine-rich repeat domain containing immune receptors and SA-induced transcription regulators are critical for functional PCD and SAR responses, respectively. By placing these recent findings in context of knowledge gained in other eukaryotic model species, we highlight potential alternative roles for processive ubiquitination in regulating the activity of SA-mediated immune responses. [ABSTRACT FROM AUTHOR]

Key message: HRE1α shows transcriptional activation activity in its C-terminal region via GCC box but not DRE/CRT and plays an important role in root development via root meristem cell division regulation. Abstract: AtERF73/HRE1 protein, a member of the Arabidopsis AP2/ERF family, contains a conserved AP2/ERF DNA-binding domain. Here, we studied the molecular function of HRE1α, a splicing variant of AtERF73/HRE1, as well as its role in root development. HRE1α-overexpressing transgenic plants (OXs) showed tolerance to submergence. HRE1α showed transcriptional activation activity via GCC box but not DRE/CRT. The 121-211 aa region of HRE1α was responsible for the transcriptional activation activity, and the region was conserved among homologs of other species but was not found in other Arabidopsis proteins. HRE1α OXs showed increased primary root length due to elevated root cell division. Our results suggest that HRE1α functions as a transcription activator in the nucleus, and plays an important role in root development through regulation of root meristem cell division. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests that homeodomain–leucine zipper I (HD-Zip) I transcription factors play important roles in abiotic stress responses, but no HD-Zip I proteins have been reported in maize. Here, a drought-induced HD-Zip I gene, Zmhdz10, was isolated from maize and characterized for its role in stress responses. Real-time quantitative PCR showed that expression of Zmhdz10 was also induced by salt stress and ABA. Transient expression of Zmhdz10–green fluorescent protein (GFP) fusion proteins in onion cells showed a nuclear localization of Zmhdz10. Yeast hybrid assays demonstrated that Zmhdz10 has transactivation and DNA-binding activity in yeast cells. Overexpression of Zmhdz10 in rice led to enhanced tolerance to drought and salt stresses and increased sensitivity to ABA. Moreover, Zmhdz10 transgenic plants had lower relative electrolyte leakage (REL), lower malondialdehyde (MDA) and increased proline content relative to wild-type plants under stress conditions, which may contribute to enhanced stress tolerance. Zmhdz10 transgenic Arabidopsis plants also exhibited enhanced tolerance to drought and salt stresses that was concomitant with altered expression of stress/ABA-responsive genes, including Δ1-Pyrroline-5-carboxylate synthetase 1 (P5CS1), Responsive to dehydration 22 (RD22), Responsive to dehydration 29B (RD29B) and ABA-insensitive 1 (ABI1). Taken together, these results suggest that Zmhdz10 functions as a transcriptional regulator that can positively regulate drought and salt tolerance in plants through an ABA-dependent signaling pathway. [ABSTRACT FROM PUBLISHER]

CarD, an essential transcription regulator in Mycobacterium tuberculosis, directly interacts with the RNA polymerase (RNAP). We used a combination of in vivo and in vitro approaches to establish that CarD is a global regulator that stimulates the formation of RNAP-holoenzyme open promoter (RPo) complexes. We determined the X-ray crystal structure of Thermus thermophilus CarD, allowing us to generate a structural model of the CarD/RPo complex. On the basis of our structural and functional analyses, we propose that CarD functions by forming protein/protein and protein/DNA interactions that bridge the RNAP to the promoter DNA. CarD appears poised to interact with a DNA structure uniquely presented by the RPo: the splayed minor groove at the double-stranded/single-stranded DNA junction at the upstream edge of the transcription bubble. Thus, CarD uses an unusual mechanism for regulating transcription, sensing the DNA conformation where transcription bubble formation initiates. [ABSTRACT FROM AUTHOR]

Synthetic organophosphates (OPs) have been used as nerve agents and pesticides due to their extreme toxicity and have caused serious environmental and human health problems. Hence, effective methods for detoxification and decontamination of OPs are of great significance. Here we constructed and used a high-throughput screening (HTS) system that was based on phenolics-responsive transcription activator for directed evolution of OP-degrading enzymes. In the screening system, phenolic compounds produced from substrates by OP-degrading enzymes bind a constitutively expressed transcription factor DmpR, initiating the expression of enhanced green fluorescent protein located at the downstream of the DmpR promoter. Fluorescence intensities of host cells are proportional to the levels of phenolic compounds, enabling the screening of OP-degrading enzymes with high catalytic activities by fluorescence-activated cell sorting. Methyl parathion hydrolase from Pseudomonas sp. WBC-3 and p-nitrophenyl diphenylphosphate were used as a model enzyme and an analogue of G-type nerve agents, respectively. The utility of the screening system was demonstrated by generating a triple mutant with a 100-fold higher kcat/Km than the wild-type enzyme after three rounds of directed evolution. The contributions of individual mutations to the catalytic efficiency were elucidated by mutational and structural analyses. The DmpR-based screening system is expected to be widely used for developing OP-degrading enzymes with greater potential. [ABSTRACT FROM PUBLISHER]

Abstract: FarA is a Zn(II)2Cys6 transcription factor which upregulates genes required for growth on fatty acids in filamentous fungi like Aspergillus nidulans. FarA is also highly similar to the cutinase transcription factor CTF1α of Fusarium solani which binds to the cutinase gene promoter in this plant pathogen. This study determines whether FarA transcriptional factor also works in the regulation of genes responsible for the production of cutinase for the degradation of a biodegradable plastic, poly-(butylene succinate-co-adipate) (PBSA), in Aspergillus oryzae. The wild-type and the farA gene disruption strains were grown in minimal agar medium with emulsified PBSA, and the wild-type showed clear zone around the colonies while the disruptants did not. Western blot analysis revealed that the cutinase protein CutL1 and a hydrophobic surface binding protein such as HsbA were produced by the wild-type but not by the disruptants. In addition, the expressions of cutL1, triacylglycerol lipase (tglA), and mono- and di-acylglycerol lipase (mdlB) genes as well as the hsbA gene were significantly lower in the disruptants compared to the wild-type. These results indicated that the FarA transcriptional factor would be implicated in the expression of cutL1 and hsbA genes that are required for the degradation of PBSA as well as lipolytic genes such as mdlB and tglA for lipid hydrolysis. [Copyright &y& Elsevier]

An important assumption of our current understanding of the mechanisms of carcinogenesis has been the belief that clarification of the cancer process would inevitably reveal some of the crucial mechanisms of normal human gene regulation. Since the momentous work of Bishop and Varmus, both the molecular and the biochemical processes underlying the events in the development of cancer have become increasingly clear. The identification of cellular signaling pathways and the role of protein kinases in the events leading to gene activation have been critical to our understanding not only of normal cellular gene control mechanisms, but also have clarified some of the important molecular and biochemical events occurring within a cancer cell. We now know that oncogenes are dysfunctional proto-oncogenes and that dysfunctional tumor suppressor genes contribute to the cancer process. Furthermore, Weinstein and others have hypothesized the phenomenon of oncogene addiction as a distinct characteristic of the malignant cell. It can be assumed that cancer cells, indeed, become dependent on such vital oncogenes. The products of these vital oncogenes, such as c-myc, may well be the Achilles heel by which targeted molecular therapy may lead to truly personalized cancer therapy. The remaining problem is the need to introduce relevant molecular diagnostic tests such as genome microarray analysis and proteomic methods, especially protein kinase identification arrays, for each individual patient. Genome wide association studies on cancers with gene analysis of single nucleotide and other mutations in functional proto-oncogenes will, hopefully, identify dysfunctional proto-oncogenes and allow the development of more specific targeted drugs directed against the protein products of these vital oncogenes. In 1984 Willis proposed a molecular and biochemical model for eukaryotic gene regulation suggesting how proto-oncogenes might function within the normal cell. That model predicted the existence of vital oncogenes and can now be used to hypothesize the biochemical and molecular mechanisms that drive the processes leading to disruption of the gene regulatory machinery, resulting in the transformation of normal cells into cancer. [ABSTRACT FROM AUTHOR]

2-oxogluatarate (2-OG), a metabolite of the highly conserved Krebs cycle, not only plays a critical role in metabolism, but also constitutes a signaling molecule in a variety of organisms ranging from bacteria to plants and animals. In cyanobacteria, the accumulation of 2-OG constitutes the signal of nitrogen starvation and NtcA, a global transcription factor, has been proposed as a putative receptor for 2-OG. Here we present three crystal structures of NtcA from the cyanobacterium Anabaena: the apoform, and two ligand-bound forms in complex with either 2-OG or its analogue 2,2-difluoropentanedioic acid. All structures assemble as homodimers, with each subunit composed of an N-terminal effector-binding domain and a C-terminal DNA-binding domain connected by a long helix (C-helix). The 2-OG binds to the effector-binding domain at a pocket similar to that used by cAMP in catabolite activator protein, but with a different pattern. Comparative structural analysis reveals a putative signal transmission route upon 2-OG binding. A tighter coiled-coil conformation of the two C-helices induced by 2-OG is crucial to maintain the proper distance between the two F-helices for DNA recognition. Whereas catabolite activator protein adopts a transition from off-to-on state upon CAMP binding, our structural analysis explains well why NtcA can bind to DNA even in its apoform, and how 2-OG just enhances the DNA-binding activity of NtcA. These.findings provided the structural insights into the function of a global transcription factor regulated by 2-0G. a metabolite standing at a crossroad between carbon and nitrogen metabolisms. [ABSTRACT FROM AUTHOR]

The broad-spectrum organophosphate insecticide chlorpyrifos (CPF)-inducible locus, chpAB, was identified on the endogenous plasmid pSymB in Sinorhizobium meliloti. The S. meliloti chpA promoter was highly induced by CPF and was induced at much lower levels by diazinon and ethion. Transcription of chpA was dependent on chpR, a CadC family transcriptional regulator located upstream of, and divergently transcribed from, chpAB. ChpR was able to mediate the CPF-inducible expression of the S. melilotichpA promoter in Escherichia coli through direct interaction with the chpAB promoter. The chpR-chpA intergenic regions of several bacterial chpRAB operons were aligned and a putative ChpR-binding sequence was proposed. Both the ChpR transcription factor and chpA promoter constitute a good candidate system for genetic-based biosensor development. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Precise modulation of transcription plays a vital role in both development and the response of all higher organisms to their environment. Temporal activation or repression of specific genes is accomplished via a plethora of transcriptional regulators. However, relatively little is known about how the activities of these proteins are controlled. Recent findings indicate that post-translational modifications fine-tune the function of transcription regulators by affecting their localization, conformation or stability. Here, we discuss these regulatory mechanisms in the context of the plant immune response. This system lends itself particularly well to studies of transcriptional regulators as activation of plant immunity is associated with rapid and dramatic reprogramming of the transcriptome. A case study of the plant immune coactivator NPR1 (nonexpressor of pathogenesis-related ( PR) genes 1) illustrates that transcription regulator activity may be controlled by redox-based modifications of cysteine thiols (e.g. disulphide bonding and S-nitrosylation), phosphorylation, and ubiquitinylation coupled to protein degradation. Importantly, cross-talk between distinct protein modifications may determine the spatial and temporal activity of transcription regulators that in turn profile the cellular transcriptome. [ABSTRACT FROM AUTHOR]

A pathogen-inducible ethylene-responsive factor gene, EREB1, was isolated and characterized in cotton ( Gossypium barbadense L.). The deduced amino acid sequence of EREB1 had some typical features of transcription factors including nuclear localization signals, an acidic activation domain, and a conserved DNA binding domain. As a single copy gene in the cotton genome, the level of expression of EREB1 in roots and leaves was significantly elevated following treatment with Verticillium wilt toxin. Presence of a GCC box-binding ability motif indicated that the EREB1 protein was capable of binding to GCC box elements in vitro as a transcriptional activator. In tobacco, overexpression of EREB1 activated pathogenesis-related genes under normal growth conditions. These results suggested that EREB1 might play an important role in biotic stress signal transduction pathways and that the overexpression of EREB1 might serve as a viable approach to enhance disease resistance t in cotton. [ABSTRACT FROM AUTHOR]

The binding of cAMP to the Escherichia coli catabolite gene activator protein (CAP) produces a conformational change that enables it to bind specific DNA sequences and regulate transcription, which it cannot do in the absence of the nucleotide. The crystal structures of the unliganded CAP containing a D138L mutation and the unliganded WT CAP were determined at 2.3 and 3.6 Å resolution, respectively, and reveal that the two DNA binding domains have dimerized into one rigid body and their two DNA recognition helices become buried. The WT structure shows multiple orientations of this rigid body relative to the nucleotide binding domain supporting earlier biochemical data suggesting that the inactive form exists in an equilibrium among different conformations. Comparison of the structures of the liganded and unliganded CAP suggests that cAMP stabilizes the active DNA binding conformation of CAP through the interactions that the N6 of the adenosine makes with the C-helices. These interactions are associated with the reorientation and elongation of the C-helices that precludes the formation of the inactive structure. [ABSTRACT FROM AUTHOR]

Photosynthesis is the primary life process in nature, and how to improve photosynthetic capacity under abiotic stresses is crucial to carbon fixation and plant productivity. As a multi-functional tree species, the leaves of Cyclocarya paliurus possess antihypertensive and hypoglycemic activities. However, the regulatory mechanism involved in the photosynthetic process of C. paliurus exposed to salinity has not yet been elucidated. In this study, the photosynthetic characteristics of C. paliurus seedlings, such as photosynthetic rate (Pn), stomatal conductance (Gs), and electron transfer rate (ETR), were investigated under different salt concentrations, while the metabolome and transcriptome analyses were conducted to unravel its molecular regulatory mechanisms. Salt stress not only significantly affected photosynthetic characteristics of C. paliurus seedlings, but also severely modified the abundance of metabolites (such as fumaric acid, sedoheptulose-7-phosphate, d-fructose-1,6-bisphosphate, and 3-phospho-d-glyceroyl phosphate) involved in central carbon metabolism, and the expression of photosynthetic genes. Through the co-expression network analysis, a total of 27 transcription factors (including ERFs, IDD, DOF, MYB, RAP) were identified to regulate photosynthetic genes under salt stress. Our findings preliminarily clarify the molecular regulatory network involved in the photosynthetic process of C. paliurus under salt stress and would drive progress in improving the photosynthetic capacity and productivity of C. paliurus by molecular technology. [ABSTRACT FROM AUTHOR]

Thinopyrum intermedium is resistant to many different pathogens. To understand the roles of ethylene response factors (ERFs) in defence responses, the first member of the ERF family in T. intermedium, TiERF1, was characterized and functionally analysed in this study. The TiERF1 gene encodes a putative protein of 292 amino acids, belonging to the B3 subgroup of the ERF transcription factor family. Biochemical assays demonstrated that the TiERF1 protein is capable of binding to the GCC box, a cis-element present in the promoters of pathogenesis-related (PR) genes, and possessing transactivation activity, as well as localizing to the nucleus. The transcript of TiERF1 in T. intermedium is rapidly induced by infection with Rhizoctonia cerealis, Fusarium graminearum, or Blumeria graminis, and ethylene, jasmonic acid, and salicylic acid treatments. More importantly, the ectopic expression of TiERF1 in tobacco activated the transcript of the PR genes of tobacco with a GCC box cis-element, and ACO and ACS genes key to ethylene synthesis, and in turn improved the resistance level to Alternaria alternata and tobacco mosaic virus, as well as causing some phenotypic changes associated with ethylene response in the transgenic tobacco plants. Taken together, TiERF1 protein as an ERF transcription activator positively regulates defence responses via the activation of some defence-related genes. [ABSTRACT FROM PUBLISHER]

In this study, a pathogen-inducible ERF (ethylene-response factor) gene in wheat, designated TaERF3, was isolated and characterized in detail. The sequence of the TaERF3 protein possesses all of the traits commonly associated with ERFs, but its entire sequence shares low identity with other ERFs of transcription factor families. The results of assays on subcelluar localization, GCC box-binding ability, and transactivation activity indicated that TaERF3 is a nuclear targeting protein and functions as a GCC box-binding transcriptional activator. Following infection with Blumeria graminis, the induction peak of TaERF3 expression occurring at 12 h in the resistant line was about six times higher than that in its susceptible parent. Following infections with Fusarium graminearum or Rhizoctonia cerealis, the TaERF3 maximum inductions in the susceptible line occurring at 12 h were about three or six times higher than those in the resistant lines, whereas after 24 h or 48 h, the transcript inductions in the resistant lines were much higher than that in the susceptible line. Furthermore, the TaERF3 transcript peak induced by salicylic acid (SA) treatment occurred at 4 h, whereas the peaks induced by exogenous ethylene and methyl jasmonate (MeJA) occurred at 24 h, all of which were earlier than those induced by pathogens in the resistant lines. These results suggested that TaERF3 might be mainly involved in the active defence response to B. graminis at an earlier stage through SA signalling, and to F. graminearum and R. cerealis at a later stage through the ethylene/jasmonic acid signalling pathways. [ABSTRACT FROM PUBLISHER]

The ZFHD recognition sequence (ZFHDRS) and NAC recognition sequence (NACRS) play an important role in the dehydration-inducible expression of the Arabidopsis thaliana EARLY RESPONSIVE TO DEHYDRATION STRESS 1 ( ERD1) gene. Using the yeast one-hybrid system, we isolated a cDNA encoding the ZFHD1 transcriptional activator that specifically binds to the 62 bp promoter region of ERD1, which contains the ZFHDRS. Both in vitro and in vivo analyses confirmed specific binding of the ZFHD1 to ZFHDRS, and the expression of ZFHD1 was induced by drought, high salinity and abscisic acid. The DNA-binding and activation domains of ZFHD1 were localized on the C-terminal homeodomain and N-terminal zinc finger domain, respectively. Microarray analysis of transgenic plants over-expressing ZFHD1 revealed that several stress-inducible genes were upregulated in the transgenic plants. Transgenic plants exhibited a smaller morphological phenotype and had a significant improvement of drought stress tolerance. Using the yeast two-hybrid system, we detected an interaction between ZFHD1 and NACRS-binding NAC proteins. Moreover, co-over-expression of the ZFHD1 and NAC genes restored the morphological phenotype of the transgenic plants to a near wild-type state and enhanced expression of ERD1 in both Arabidopsis T87 protoplasts and transgenic Arabidopsis plants. [ABSTRACT FROM AUTHOR]

To identify artificial DNA segments that can stably express transgenes in the genome of host cells, we built a series of curved DNA segments that mimic a left-handed superhelical structure. Curved DNA segments of 288 bp (T32) and 180 bp (T20) were able to activate transcription from the herpes simplex virus thymidine kinase ( tk) promoter by approximately 150-fold and 70-fold, respectively, compared to a control in a transient transfection assay in COS-7 cells. The T20 segment was also able to activate transcription from the human adenovirus type 2 E1A promoter with an 18-fold increase in the same assay system, and also activated transcription from the tk promoter on episomes in COS-7 cells. We also established five HeLa cell lines with genomes containing T20 upstream of the transgene promoter and control cell lines with T20 deleted from the transgene locus. Interestingly, T20 was found to activate transcription in all the stable transformants, irrespective of the locus. This suggests that the T20 segment may allow stable expression of transgenes, which is of importance in many fields, and may also be useful for the construction of nonviral vectors for gene therapy. [ABSTRACT FROM AUTHOR]

The temperate coliphage λ, after infecting its host bacterium Escherichia coli, can develop either along the lytic or the lysogenic pathway. Crucial to the lysis/lysogeny decision is the homotetrameric transcription-activator protein CII (4 × 11 kDa) of the phage that binds to a unique direct-repeat sequence T-T-G-C-N6-T-T-G-C at each of the three phage promoters it activates: pE, pI and paQ. Several regions of CII have been identified for its various functions (DNA binding, oligomerization, and susceptibility to host protease), but the crystal structure of the protein long remained elusive. Here, we present the three-dimensional structure of CII at 2.6-Å resolution. The CII monomer is comprised of four α helices and a disordered C terminus. The first three helices (α1-α3) form a compact domain, whereas the fourth helix (α4) protrudes in different orientations in each subunit A four-helix bundle, formed by α4 from each subunit holds the tetramer. The quaternary structure can be described as a dimer of dimers, but the tetramer does not exhibit a closed symmetry. This unusual quaternary arrangement allows the placement of the helix-turn-helix motifs of two of the four CII subunits for interaction with successive major grooves of B-DNA, from one face of DNA. This structure provides a simple explanation for how a homotetrameric protein may recognize a direct-repeat DNA sequence rather than the inverted-repeat sequences of most prokaryotic activators. [ABSTRACT FROM AUTHOR]

MalT, the dedicated transcriptional activator of the maltose regulon in Escherichia coli, is subject to multiple controls. Maltotriose, the inducer, promotes MalT self-association, a critical step in promoter binding, whereas three proteins acting as negative allosteric effectors (MalK, the ABC-component of the maltodextrin transporter, MalY, and Aes) antagonize maltotriose binding. All of these regulatory signals are integrated by a novel signal transduction module that comprises three out of the four MalT structural domains: DT1, the ATP-binding domain that contains determinants recognized by the negative effectors, DT2, and DT3, the maltotriose-binding domain. For a better insight into the role of DT3 in signal integration, we PCR mutagenized the DT3-encoding region and screened for gain of function mutations in a malK + strain in the absence of repression by MalY or Aes. Most of the mutations isolated alter one of seven residues that are located in DT3 helices 10 and 11, or in the turn between them and delineate a surface-exposed motif. In vivo and in vitro analyses revealed that the substitutions altering the so-called H10/H11 motif do not affect the ability of MalT to activate transcription or its sensitivity to MalY and Aes, but dramatically decrease its sensitivity to MalK. We propose that MalT/MalK interaction might involve two distinct contact sites on each partner. These sites would be located in DT1 and DT3 of MalT, and in the nucleotide-binding domain and the regulatory domain of MalK. Such a two-point interaction model would explain how the regulatory activity of MalK might be coupled to transport. [Copyright &y& Elsevier]

MalT, the dedicated transcriptional activator of the maltose regulon in Escherichia coli, is the prototype for a family of large (∼100kDa) transcriptional activators. MalT self-association plays a key role in recognition of the target promoters, which contain several MalT sites that are cooperatively bound by the activator. The unliganded form of MalT is monomeric. The protein self-associates only in the presence of both ATP (or AMP-PNP, a non-hydrolysable analog of ATP) and maltotriose, the inducer. Here, we report cryo-electron microscopy analyses of MalT multimeric forms. We show that, in the presence of maltotriose and AMP-PNP, MalT associates into novel, polydisperse, curved homopolymers. The building block, corresponding to a MalT monomer, comprises an outer globular domain connected by a peduncle to an inner domain that mediates self-association. Image analyses highlight the significant conformational flexibility of these polymeric forms. In the presence of a DNA fragment containing a MalT-controlled promoter, malPp500, MalT forms homopolymers with a much smaller radius of curvature and a different conformation. We propose that MalT binding to the target promoters involves the assembly of a MalT homo-oligomer that is governed by the array of MalT sites present. [Copyright &y& Elsevier]

The SoxR protein of Escherichia coli responds to redox signals by activating the transcription of soxS, which encodes another transcription activator that directly stimulates oxidative stress genes. In contrast, Pseudomonas aeruginosa has an open reading frame (ORF) encoding a putative protein homologous to E. coli SoxR, but not to SoxS. Instead of a soxS homolog, ORFs encoding an unknown hypothetical protein and soxR are arranged divergently with their 5′ ends separated by a 78 bp region containing a sequence homologous to the SoxR-binding soxS promoter. In this study, we report the overproduction and purification of SoxR from P. aeruginosa to investigate the mechanism of gene activation by SoxR. The spectroscopic properties of the purified SoxR protein indicate that it contains a redox active iron-sulfur [2Fe-2S] cluster. Redox titration of the SoxR protein revealed a midpoint potential of –290 mV. The SoxR protein specifically binds a fragment of the SoxS promoter–like region in a concentration-dependent fashion, as shown by both gel mobility shift and fluorescence polarization assays. The purified SoxR stimulates the in vitro transcription of the gene encoding the hypothetical protein in P. aeruginosa. This activity was lost following reduction of the SoxR [2Fe-2S] clusters. The levels of mRNA in the hypothetical protein increased in paraquat-treated cells. These results indicate that P. aeruginosa SoxR is a direct transcriptional activator of the hypothetical protein, and suggest that SoxR proteins may play multiple regulatory roles as a transcription factor in addition to its protective role in oxidative stress. [ABSTRACT FROM PUBLISHER]

j. liu, s.-y. sun and t.-h. wang. 2004. To construct a yeast one-hybrid system and isolate transcriptional activators. A 1·1-kb promoter region of xylanase2 from Trichoderma reesei was cloned by PCR and sequenced (GenBank accession number: ). Sequence analysis revealed that typical binding sites for several transcription factors in filamentous fungi, such as CREI, XLNR, ALCR, AREA and CCAAT enhancer, are located in the promoter. To isolate xyn2 transcription factors, the reporter plasmid of a yeast one-hybrid system was constructed on the backbone of the plasmid pRS415 containing the leu2 selective marker, with the xyn2 promoter region and Saccharomyces cerevisiae his4 as a reporter gene. The reporter gene contained 123-bp minimal promoter region. The S. cerevisiae H158 strain containing the reporter plasmid was transformed with a T. reesei expression cDNA library, and 34 transformants were collected from SC-Leu-His-Ura plates. The isolation of the gene ace2 from several transformants showed that the one-hybrid system approach was successful. Then, approx. 59 mg l−1 of ace2 was overexpressed in Escherichia coli BL21. The yeast one-hybrid system is suitable for isolating transcription factors of filamentous fungi. ACE II is a main and universal transcriptional activator that controls cellulase and hemicellulase transcription regulation in T. reesei. [ABSTRACT FROM AUTHOR]

The centromere binding factor 1 (Cbf1) is necessary for proper chromosome segregation and transcriptional activation of methionine biosynthesis genes in the yeast Saccharomyces cerevisiae and is essential for viability in the related yeasts Kluyveromyces lactis and Candida glabrata. To study the function of Cbf1p in Candida albicans, the major human fungal pathogen, we constructed strains in which both alleles of the CaCBF1 gene were deleted. The Δcbf1 mutants exhibited a slow growth phenotype and were temperature-sensitive at 42 °C. In addition, the mutants were auxotrophic for sulfur amino acids and could grow on minimal medium only when it was supplemented with either methionine or cysteine, suggesting that CaCBF1 is necessary for the expression of genes involved in assimilation of inorganic sulfate. Deletion of CaCBF1 also resulted in morphological abnormalities, many cells being unusually large. All mutant phenotypes were complemented by reintroduction of a functional CaCBF1 copy. The Δcbf1 mutants neither showed enhanced sensitivity to the microtubule destabilizing agent thiabendazole nor did they exhibit an increased frequency of chromosome loss. These results suggest that Cbf1p is not necessary for efficient chromosome segregation in C. albicans. [Copyright &y& Elsevier]