Electrophoretic Gel Mobility Shift Assays Research Articles

Phosphate effect on filipin production and morphological differentiation in Streptomyces filipinensis and the role of the PhoP transcription factor.

The biosynthesis of the antifungal filipin in Streptomyces filipinensis is very sensitive to phosphate regulation. Concentrations as low as 2.5 mM block filipin production. This effect is, at least in part, produced by repression of the transcription of most filipin biosynthetic genes. The role of the two-component PhoRP system in this process was investigated. The phoRP system of S. filipinensis was cloned and transcriptionally characterised. PhoP binds to two PHO boxes present in one of its two promoters. Filipin production was greatly increased in ΔphoP and ΔphoRP mutants, in agreement with a higher transcription of the fil genes, and the effect of phosphate repression on the antibiotic production of these strains was significantly reduced. No PhoP binding was observed by electrophoretic mobility gel shift assays (EMSAs) with the promoter regions of the fil gene cluster thus suggesting an indirect effect of mutations. Binding assays with cell-free extracts from the wild-type and mutant strains on fil genes promoters revealed retardation bands in the parental strain that were absent in the mutants, thus suggesting that binding of the putative transcriptional regulator or regulators controlled by PhoP was PhoP dependent. Noteworthy, PhoP or PhoRP deletion also produced a dramatic decrease in sporulation ability, thus indicating a clear relationship between the phosphate starvation response mediated by PhoP and the sporulation process in S. filipinensis. This effect was overcome upon gene complementation, but also by phosphate addition, thus suggesting that alternative pathways take control in the absence of PhoRP.

Structural insights into methylated DNA recognition by the C-terminal zinc fingers of the DNA reader protein ZBTB38

Methyl-CpG-binding proteins (MBPs) are selective readers of DNA methylation that play an essential role in mediating cellular transcription processes in both normal and diseased cells. This physiological function of MBPs has generated significant interest in understanding the mechanisms by which these proteins read and interpret DNA methylation signals. Zinc finger and BTB domain-containing 38 (ZBTB38) represents one member of the zinc finger (ZF) family of MBPs. We recently demonstrated that the C-terminal ZFs of ZBTB38 exhibit methyl-selective DNA binding within the ((A/G)TmCG(G/A)(mC/T)(G/A)) context both in vitro and within cells. Here we report the crystal structure of the first four C-terminal ZBTB38 ZFs (ZFs 6-9) in complex with the previously identified methylated consensus sequence at 1.75 Å resolution. From the structure, methyl-selective binding is preferentially localized at the 5' mCpG site of the bound DNA, which is facilitated through a series of base-specific interactions from residues within the α-helices of ZF7 and ZF8. ZF6 and ZF9 primarily stabilize ZF7 and ZF8 to facilitate the core base-specific interactions. Further structural and biochemical analyses, including solution NMR spectroscopy and electrophoretic mobility gel shift assays, revealed that the C-terminal ZFs of ZBTB38 utilize an alternative mode of mCpG recognition from the ZF MBPs structurally evaluated to date. Combined, these findings provide insight into the mechanism by which this ZF domain of ZBTB38 selectively recognizes methylated CpG sites and expands our understanding of how ZF-containing proteins can interpret this essential epigenetic mark.

Molecular determinants of Burkholderia pseudomallei BpeEF-OprC efflux pump expression

Burkholderia pseudomallei, the cause of melioidosis, is intrinsically resistant to many antibiotics. Acquired multidrug resistance, including resistance to doxycycline and co-trimoxazole used for melioidosis eradication phase therapy, is mainly attributed to constitutive expression of the BpeEF-OprC efflux pump. Constitutive expression of this pump is caused by mutations affecting two highly similar LysR-type transcriptional regulators (LTTR), BpeT and BpeS, but their interaction with the regulatory region governing BpeEF-OprC expression has not yet been studied. The bpeE-bpeF-oprC genes are distally located in the llpE-bpeE-bpeF-oprC operon. The llpE gene encodes a putative lipase/esterase of unknown function. We show that in a bpeT mutant llpE is constitutively co-transcribed with bpeE-bpeF-oprC. As expected from previous studies with B. cenocepacia, deletion of llpE does not affect antibiotic efflux. Using transcriptional bpeE'-lacZ fusions, we demonstrate that the 188 bp bpeT-llpE intergenic region located between bpeT and the llpE-bpeE-bpeF-oprC operon contains regulatory elements needed for control of bpeT and llpE-bpeE-bpeF-oprC operon expression. By native polyacrylamide gel electrophoresis and electrophoretic mobility shift assays with purified recombinant BpeT and BpeS proteins, we show BpeT and BpeS form oligomers that share a 14 bp binding site overlapping the essential region required for llpE-bpeE-bpeF-oprC expression. The binding site contains the conserved T-N11-A LTTR box motif involved in binding of LysR proteins, which in concert with two other possible LTTR boxes may mediate BpeT and BpeS regulation of BpeEF-OprC expression. These studies form the basis for further investigation of BpeEF-OprC expression and regulation at the molecular level by yet unknown external stimuli.

Characterization of Small Molecules Inhibiting the Pro-Angiogenic Activity of the Zinc Finger Transcription Factor Vezf1.

Discovery of inhibitors for endothelial-related transcription factors can contribute to the development of anti-angiogenic therapies that treat various diseases, including cancer. The role of transcription factor Vezf1 in vascular development and regulation of angiogenesis has been defined by several earlier studies. Through construction of a computational model for Vezf1, work here has identified a novel small molecule drug capable of inhibiting Vezf1 from binding to its cognate DNA binding site. Using structure-based design and virtual screening of the NCI Diversity Compound Library, 12 shortlisted compounds were tested for their ability to interfere with the binding of Vezf1 to DNA using electrophoretic gel mobility shift assays. We identified one compound, T4, which has an IC50 of 20 μM. Using murine endothelial cells, MSS31, we tested the effect of T4 on endothelial cell viability and angiogenesis by using tube formation assay. Our data show that addition of T4 in cell culture medium does not affect cell viability at concentrations lower or equal to its IC 50 but strongly inhibits the network formation by MSS31 in the tube formation assays. Given its potential efficacy, this inhibitor has significant therapeutic potential in several human diseases.

Induction of apoptosis by Bigelovii A through inhibition of NF‑κB activity.

Bigelovii A is a 30-nortriterpenoid glycoside, isolated from Salicornia bigelovii Torr. Until now, the effect of Bigelovii A on breast cancer treatment was unknown. The present research indicated that Bigelovii A significantly inhibited the proliferation of human breast cancer cells (MCF-7, MDA-MB-231 and MDA-MB-468) in a concentration-dependent manner. It was particularly effective in MCF7 cells, with an IC50 value of 4.10±1.19 µM. The anti-proliferative effect of Bigelovii A was ascribed to the induction of apoptosis, which was characterized by chromatin condensation, externalization of phosphatidylserine on the plasma membrane, hypodiploid DNA, activation of caspases and poly (ADP-ribose) polymerase cleavage. Furthermore, Bigelovii A reduced B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl) expression and caused disruption of mitochondrial membrane potential, which are indicative features of mitochondria-dependent apoptotic signals. It was also identified that Bigelovii A downregulated the constitutive activation of nuclear factor (NF)-κB, as indicated by the electrophoretic mobility gel shift assay and immunocytochemistry. Furthermore, Bigelovii A suppressed constitutive IκBα phosphorylation via inhibition of IκB kinase activity. In addition to the effects on Bcl-2 and Bcl-xl, Bigelovii A also downregulated the expression of the NF-κB-regulated gene products, Cyclin D1 and cyclooxygenase-2. This led to the induction of apoptosis and arrest of cells at the G1 phase of the cell cycle.

Photoactive platinum(II) complexes of nonsteroidal anti-inflammatory drug naproxen: Interaction with biological targets, antioxidant activity and cytotoxicity

The effect on the therapeutic efficacy of Pt(II) complexes on combining non-steroidal anti-inflammatory drugs (NSAIDs) is an attractive strategy to circumvent chronic inflammation mediated by cancer and metastasis. Two square-planar platinum(II) complexes: [Pt(dach)(nap)Cl] (1) and [Pt(dach)(nap)2] (2), where dach = (1R,2R)-dichloro(cyclohexane-1,2-diamine) and NSAID drug naproxen (nap), have been designed for studying their biological activity. The naproxen bound to the Pt(II) centre get released upon photoirradiation with low-power UV-A light as confirmed by the significant enhancement in emission intensities of the complexes. The compounds were evaluated for their photophysical properties, photostability, reactivity with 5′-guanosine monophophosphate (5′-GMP), interactions with CT-DNA and BSA, antioxidant activity and reactive oxygen species mediated photo-induced DNA damage properties. ESI-MS studies demonstrated the formation of bis-adduct with 5′-GMP and the formation of PtII-DNA crosslinks by gel electrophoretic mobility shift assay and ITC studies. The interaction of the complexes 1 and 2 with the CT-DNA exhibits potential binding affinity (Kb ∼ 104 M−1, Kapp∼ 105 M−1), implying intercalation to CT-DNA through planar naphthyl ring of the complexes. Both the complexes also exhibit strong binding affinity towards BSA (KBSA∼ 105 M−1). The complexes exhibit efficient DNA damage activity on irradiation at 365 nm via formation of singlet oxygen (1O2) and hydroxyl radical (•OH) under physiological conditions. Both the complexes were cytotoxic in dark and exhibit significant enhancement of cytotoxicity upon photo-exposure against HeLa and HepG2 cancer cells giving IC50 values ranging from 8 to 12 μM for 1 and 2. The cellular internalization data showed cytosolic and nuclear localization of the complexes in the HeLa cells.

Crotonis Fructus Extract Inhibits 12-O-Tetradecanoylphorbol-13-Acetate-Induced Expression of Matrix Metalloproteinase-9 via the Activator Protein-1 Pathway in MCF-7 Cells.

PurposeMetastatic cancers spread from the primary site of origin to other parts of the body. Matrix metalloproteinase-9 (MMP-9) is essential in metastatic cancers owing to its major role in cancer cell invasion. Crotonis fructus (CF), the mature fruits of Croton tiglium L., have been used for the treatment of gastrointestinal disturbance in Asia. In this study, the effect of the ethanol extract of CF (CFE) on MMP-9 activity and the invasion of 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated MCF-7 cells was examined.MethodsThe cell viability was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The expression of MMP-9 was examined by Western blotting, zymography, and real-time polymerase chain reaction. An electrophoretic mobility gel shift assay was performed to detect activator protein-1 (AP-1) DNA binding activity and cell invasiveness was measured by an in vitro Matrigel invasion assay.ResultsCFE significantly suppressed MMP-9 expression and activation in a dose-dependent manner. Furthermore, CFE attenuated the TPA-induced activation of AP-1.ConclusionThe results indicated that the inhibitory effects of CFE against TPA-induced MMP-9 expression and MCF-7 cell invasion were dependent on the protein kinase C δ/p38/c-Jun N-terminal kinase/AP-1 pathway. Therefore, CFE could restrict breast cancer invasiveness owing to its ability to inhibit MMP-9 activity.

Epigallocatechin-3-gallate alleviates paraquat-induced acute lung injury and inhibits upregulation of toll-like receptors

AimsTo evaluate the detoxifying effect of epigallocatechin-3-gallate (EGCG) on paraquat (PQ)-induced acute lung injury in mice, and to explore the action mechanisms. Main methodsFollowing administration of PQ, the mice received a low, a medium or a high dose of EGCG daily for three days. Histopathology of the lungs were examined by H&E staining. The levels of inflammatory cytokines, such as TNF-α, IL-1β and IL-6, in the bronchoalveolar lavage fluid were measured by enzyme-linked immunosorbent assay. Activation of NF-κB was assessed by Western blot and electrophoretic mobility gel shift assay. The expression of toll-like receptor (TLR)-2, 4, 9 and TLR adaptors (MyD88 and TRAF6) was detected by Western blot and immunohistochemical staining. The protective effect of EGCG against PQ toxicity was validated in vitro using A549 lung cancer cell line. Key findingsTreatment with EGCG dose-dependently attenuated PQ-induced acute lung injury in mice by reducing alveolar edema, hemorrhage, inflammatory cell infiltration and production of inflammatory cytokines. EGCG inhibited the activation of NF-κB and the upregulation of TLR 2, 4 and 9 as well as their adaptors MyD88 and TRAF6 in the lungs following PQ challenge. In addition, EGCG significantly reduced PQ-induced cell death, cytokine production, activation of NF-κB, and upregulation of TLRs and adaptors in A549 cells. SignificanceOur data suggest that TLR-mediated activation of NF-κB in the non-immune pulmonary cells could be involved in PQ-induced acute lung injury, and it may serve as a target of EGCG against PQ pulmonary toxicity.

SlnR is a positive pathway-specific regulator for salinomycin biosynthesis in Streptomyces albus.

Salinomycin, a polyether antibiotic produced by Streptomyces albus, is widely used in animal husbandry as an anticoccidial drug and growth promoter. Situated within the salinomycin biosynthetic gene cluster, slnR encodes a LAL-family transcriptional regulator. The role of slnR in salinomycin production in S. albus was investigated by gene deletion, complementation, and overexpression. Gene replacement of slnR from S. albus chromosome results in almost loss of salinomycin production. Complementation of slnR restored salinomycin production, suggesting that SlnR is a positive regulator of salinomycin biosynthesis. Overexpression of slnR in S. albus led to about 25% increase in salinomycin production compared to wild type. Quantitative RT-PCR analysis revealed that the expression of most sal structural genes was downregulated in the ΔslnR mutant but upregulated in the slnR overexpression strain. Electrophoretic mobility gel shift assays (EMSAs) also revealed that SlnRDBD binds directly to the three intergenic regions of slnQ-slnA1, slnF-slnT1, and slnC-slnB3. The SlnR binding sites within the three intergenic regions were determined by footprinting analysis and identified a consensus-directed repeat sequence 5'-ACCCCT-3'. These results indicated that SlnR modulated salinomycin biosynthesis as an enhancer via interaction with the promoters of slnA1, slnQ, slnF, slnT1, slnC, and slnB3 and activates the transcription of most of the genes belonging to the salinomycin gene cluster but not its own transcription.

Expression of wnt4/5 during reproductive cycle of catfish and wnt5 promoter analysis.

Signaling molecules, Wnt4 and Wnt5, are essential for ovarian growth during developmental stages in mammals. Although these molecules were identified in several teleosts, their precise expression and role in reproductive processes have not yet been explored in any lower vertebrates. In view of this, using catfish, Clarias batrachus as an animal model, cloning and expression analysis of wnt4 and wnt5 were analyzed in different tissues, at various developmental stages, during ovarian reproductive cycle and after gonadotropin induction. These studies indicate a plausible influence of Wnts in ovarian development and recrudescence. Transcript and protein localization revealed their presence in peri-nucleolar, pre-vitellogenic, vitellogenic and follicular layer of post-vitellogenic oocytes. Synchronous expression of pax2 and wnt5 during the ovarian development and recrudescence of catfish led us to analyze the importance of putative binding element of Pax2 in the 5'-promoter motif of wnt5 Promoter activity of wnt5 was analyzed by luciferase assays after transfecting progressive deletion constructs in pGL3 basic vector into the mammalian cell lines (HEK 293 and CHO). The constructs having putative Pax2 motif showed high promoter activity compared with controls. Likewise, the constructs with site-directed mutagenesis showed increased activity after supplementing recombinant Pax2 indicating the prominence of this motif in wnt5 promoter, in vitro Electrophoretic gel mobility shift, supershift and chromatin immunoprecipitation assays confirmed the binding of Pax2 to its corresponding cis-acting element in the upstream of wnt5 This study is the first of its kind to report the critical transcriptional interaction of Pax2 on wnt5 vis-à-vis ovarian development in teleosts.

Allele-specific DNA methylation reinforces PEAR1 enhancer activity

AbstractGenetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G allele of rs12041331, an intronic cytosine guanine dinucleotide-single-nucleotide polymorphism (CpG-SNP), is associated with higher PEAR1 expression in platelets and endothelial cells than the minor A allele. The molecular mechanism underlying this difference remains elusive. We have characterized the histone modification profiles of the intronic region surrounding rs12041331 and identified H3K4Me1 enhancer-specific enrichment for the region that covers the CpG-SNP. Interestingly, methylation studies revealed that the CpG site is fully methylated in leukocytes of GG carriers. Nuclear protein extracts from megakaryocytes, endothelial cells, vs control HEK-293 cells show a 3-fold higher affinity for the methylated G allele compared with nonmethylated G or A alleles in a gel electrophoretic mobility shift assay. To understand the positive relationship between methylation and gene expression, we studied DNA methylation at 4 different loci of PEAR1 during in vitro megakaryopoiesis. During differentiation, the CpG-SNP remained fully methylated, while we observed rapid methylation increases at the CpG-island overlapping the first 5′-untranslated region exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared with GG homozygote. This CpG-island contains binding sites for the methylation-sensitive transcription factor CTCF, whose binding is known to play a role in enhancer activation and/or repression. In conclusion, we report the molecular characterization of the first platelet function-related CpG-SNP, a genetic predisposition that reinforces PEAR1 enhancer activity through allele-specific DNA methylation.

Effects of Cu(II) and cisplatin on the stability of Specific protein 1 (Sp1)-DNA binding: Insights into the regulation of copper homeostasis and platinum drug transport

The human high-affinity copper transporter 1 (hCtr1) transports both Cu(I) and cisplatin (cDDP). Because Cu deficiency is lethal yet Cu overload is poisonous, hCtr1 expression is transcriptionally upregulated in response to Cu deficiency but is downregulated under Cu replete conditions in controlling Cu homeostasis. The up- and down-regulation of hCtr1 is regulated by Specific protein 1 (Sp1), which itself is also correspondingly regulated under these Cu conditions. hCtr1 expression is also upregulated by cDDP via upregulation of Sp1. The underlying mechanisms of these regulations are unknown. Using gel-electrophoretic mobility shift assays, we demonstrated here that Sp1-DNA binding affinity is reduced under Cu replete conditions but increased under reduced Cu conditions. Similarly, Sp1-DNA binding affinity is increased by cDDP treatment. This in vitro system demonstrated, for the first time, that regulation of Sp1/hCtr1 expression by these agents is modulated by the stability of Sp1-DNA binding, the first step in the Sp1-mediated transcriptional regulation process.

Advanced purification strategy for CueR, a cysteine containing copper(I) and DNA binding protein

Metal ion regulation is essential for living organisms. In prokaryotes metal ion dependent transcriptional factors, the so-called metalloregulatory proteins play a fundamental role in controlling the concentration of metal ions. These proteins recognize metal ions with an outstanding selectivity. A detailed understanding of their function may be exploited in potential health, environmental and analytical applications. Members of the MerR protein family sense a broad range of mostly late transition and heavy metal ions through their cysteine thiolates. The air sensitivity of latter groups makes the expression and purification of such proteins challenging. Here we describe a method for the purification of the copper-regulatory CueR protein under optimized conditions. In order to avoid protein precipitation and/or eventual aggregation and to get rid of the co-purifying Escherichia coli elongation factor, our procedure consisted of four steps supplemented by DNA digestion. Subsequent anion exchange on Sepharose FF Q 16/10, affinity chromatography on Heparin FF 16/10, second anion exchange on Source 30 Q 16/13 and gel filtration on Superdex 75 26/60 resulted in large amounts of pure CueR protein without any affinity tag. Structure and functionality tests performed with mass spectrometry, circular dichroism spectroscopy and electrophoretic gel mobility shift assays approved the success of the purification procedure.

An ankylosing spondylitis-associated genetic variant in the IL23R-IL12RB2 intergenic region modulates enhancer activity and is associated with increased Th1-cell differentiation

ObjectivesTo explore the functional basis for the association between ankylosing spondylitis (AS) and single-nucleotide polymorphisms (SNPs) in the IL23R-IL12RB2 intergenic region.MethodsWe performed conditional analysis on genetic association data and used...

Interaction between TATA-Binding Protein (TBP) and Multiprotein Bridging Factor-1 (MBF1) from the Filamentous Insect Pathogenic Fungus Beauveria bassiana.

TATA-binding protein (TBP) is a ubiquitous component of eukaryotic transcription factors that acts to nucleate assembly and position pre-initiation complexes. Multiprotein bridging factor 1 (MBF1) is thought to interconnect TBP with gene specific transcriptional activators, modulating transcriptional networks in response to specific signal and developmental programs. The insect pathogen, Beauveria bassiana, is a cosmopolitan fungus found in most ecosystems where it acts as an important regulator of insect populations and can form intimate associations with certain plants. In order to gain a better understanding of the function of MBF1 in filamentous fungi, its interaction with TBP was demonstrated. The MBF1 and TBP homologs in B. bassiana were cloned and purified from a heterologous E. coli expression system. Whereas purified BbTBP was shown to be able to bind oligonucleotide sequences containing the TATA-motif (Kd ≈ 1.3 nM) including sequences derived from the promoters of the B. bassiana chitinase and protease genes. In contrast, BbMBF1 was unable to bind to these same target sequences. However, the formation of a ternary complex between BbMBF1, BbTBP, and a TATA-containing target DNA sequence was seen in agarose gel electrophoretic mobility shift assays (EMSA). These data indicate that BbMBF1 forms direct interactions with BbTBP, and that the complex is capable of binding to DNA sequences containing TATA-motifs, confirming that BbTBP can link BbMBF1 to target sequences as part of the RNA transcriptional machinery in fungi.

The genetic association of RUNX3 with ankylosing spondylitis can be explained by allele-specific effects on IRF4 recruitment that alter gene expression

ObjectivesTo identify the functional basis for the genetic association of single nucleotide polymorphisms (SNP), upstream of the RUNX3 promoter, with ankylosing spondylitis (AS).MethodsWe performed conditional analysis of genetic association data...

Regulation of arsenite oxidation by the phosphate two-component system PhoBR in Halomonas sp. HAL1.

Previously, the expression of arsenite [As(III)] oxidase genes aioBA was reported to be regulated by a three-component regulatory system, AioXSR, in a number of As(III)-oxidizing bacterial strains. However, the regulation mechanism is still unknown when aioXSR genes are absent in some As(III)-oxidizing bacterial genomes, such as in Halomonas sp. HAL1. In this study, transposon mutagenesis and gene knock-out mutation were performed, and two mutants, HAL1-phoR931 and HAL1-▵phoB, were obtained in strain HAL1. The phoR and phoB constitute a two-component system which is responsible for phosphate (Pi) acquisition and assimilation. Both of the mutants showed negative As(III)-oxidation phenotypes in low Pi condition (0.1 mM) but not under normal Pi condition (1 mM). The phoBR complementation strain HAL1-▵phoB-C reversed the mutants' null phenotypes back to wild type status. Meanwhile, lacZ reporter fusions using pCM-lacZ showed that the expression of phoBR and aioBA were both induced by As(III) but were not induced in HAL1-phoR931 and HAL1-▵phoB. Using 15 consensus Pho box sequences, a putative Pho box was found in the aioBA regulation region. PhoB was able to bind to the putative Pho box in vivo (bacterial one-hybrid detection) and in vitro (electrophoretic mobility gel shift assay), and an 18-bp binding sequence containing nine conserved bases were determined. This study provided the evidence that PhoBR regulates the expression of aioBA in Halomonas sp. HAL1 under low Pi condition. The new regulation model further implies the close metabolic connection between As and Pi.

The PhoP transcription factor negatively regulates avermectin biosynthesis in Streptomyces avermitilis.

Bacteria sense and respond to the stress of phosphate limitation, anticipating Pi deletion/starvation via the two-component PhoR-PhoP system. The role of the response regulator PhoP in primary metabolism and avermectin biosynthesis in Streptomyces avermitilis was investigated. In response to phosphate starvation, S. avermitilis PhoP, like Streptomyces coelicolor and Streptomyces lividans PhoP, activates the expression of phoRP, phoU, and pstS by binding to the PHO boxes in their promoter regions. Avermectin biosynthesis was significantly increased in ΔphoP deletion mutants. Electrophoretic mobility gel shift assay (EMSA) and DNase I footprinting assays showed that PhoP can bind to a PHO box formed by two direct repeat units of 11 nucleotides located downstream of the transcriptional start site of aveR. By negatively regulating the transcription of aveR, PhoP directly affects avermectin biosynthesis in S. avermitilis. PhoP indirectly affects melanogenesis on Casaminoacids Minimal Medium (MMC) lacking supplemental phosphate. Nitrogen metabolism and some key genes involved in morphological differentiation and antibiotic production in S. avermitilis are also under the control of PhoP.

The RNA-binding properties and domain of Rice stripe virus nucleocapsid protein.

The nucleocapsid protein (NP) of rice stripe virus (RSV) encapsidates viral genomic RNAs to form virion. The binding of NP with RNA is essential for the formation of virus particle. In this study, the binding specificity of RSV NP to RNA and the domains within the NP that mediate this interaction were investigated by gel electrophoretic mobility shift assays and Northwestern blot analysis. The results demonstrated that RSV NP was able to bind to all synthetic RNAs and DNAs without sequence specificity. Using a series of truncated NPs expressed in E. coli and synthetic peptides, we mapped the RNA-binding domain of NP to the central region from amino acid residues 201-232. Further alanine substitution analysis revealed that Lys(206), Lys(207), Lys(220), and Tyr(221) in the RNA-binding domain were essential for NP to bind with RNA.

Hypusination of eukaryotic initiation factor 5A via cAMP-PKA-ERK1/2 pathway is required for ligand-induced downregulation of LH receptor mRNA expression in the ovary

Luteinizing hormone receptor (LHR) mRNA expression in the ovary is regulated post-transcriptionally by an LH receptor mRNA binding protein (LRBP). Eukaryotic initiation factor 5A (EIF5A), identified as an LRBP-interacting protein plays a crucial role in LHR mRNA expression. In this study, we have demonstrated that during hCG-induced LHR downregulation, a significant upregulation of eIF5A mRNA expression and hypusination of eIF5A protein occurs in a time dependent manner. Pretreatment with H89, a specific inhibitor of PKA, and U0126, a specific inhibitor of ERK1/2 significantly inhibited both hCG-induced eIF5A mRNA expression and hypusination of eIF5A protein. Pretreatment with GC7, a specific inhibitor of eIF5A hypusination significantly abolished hCG-induced LRBP mRNA and protein expression. Furthermore, GC7 pretreatment significantly inhibited hCG-induced interaction of LRBP with LHR mRNA as assessed by RNA electrophoretic mobility gel shift assay (REMSA). GC7 treatment also reversed LHR mRNA downregulation. Taken together, these results suggest that hCG-induced LHR mRNA downregulation is mediated by cAMP-PKA-ERK1/2 signaling leading to activation of eIF5A hypusination.