Transition metal ion activation of DNA binding by the diphtheria tox repressor requires the formation of stable homodimers.

The diphtheria tox repressor (DtxR) is a transition metal ion-dependent regulatory element that controls the expression of diphtheria toxin and several genes involved in the synthesis of siderophores in Corynebacterium diphtheriae. In the presence of transition metal ions apo-DtxR becomes activated and specifically binds to its target DNA sequences. We demonstrate by glutaraldehyde cross-linking that monomeric apo-DtxR is in weak equilibrium with a dimeric form and that upon addition of activating metal ions to the reaction mixture a dimeric complex is stabilized. Addition of the DNA-binding-defective mutant apo-DtxR(delta 1-47) to apo-DtxR in the absence of transition metal ions inhibits conversion of the apo-repressor to its activated DNA-binding form. We also show that the binding of Ni2+ to both apo-DtxR and apo-DtxR(delta 1-47) is cooperative and that upon ion binding there is a conformational change in the environment of the indole ring moiety of Trp-104. For the wild-type repressor the consequences of this conformational change include a shift in equilibrium toward dimer formation and activation of target DNA binding by the repressor. We conclude that the formation of DtxR homodimers is mediated through a protein-protein interaction domain that is also activated on metal ion binding.

Identification of NAB1, a repressor of NGFI-A- and Krox20-mediated transcription.

NGFI-A (also called Egr1, Zif268, or Krox24) and the closely related proteins Krox20, NGFI-C, and Egr3 are zinc-finger transcription factors encoded by immediate-early genes which are induced by a wide variety of extracellular stimuli. NGFI-A has been implicated in cell proliferation, macrophage differentiation, synaptic activation, and long-term potentiation, whereas Krox20 is critical for proper hindbrain segmentation and peripheral nerve myelination. In previous work, a structure/function analysis of NGFI-A revealed a 34-aa inhibitory domain that was hypothesized to be the target of a cellular factor that represses NGFI-A transcriptional activity. Using the yeast two-hybrid system, we have isolated a cDNA clone which encodes a protein that interacts with this inhibitory domain and inhibits the ability of NGFI-A to activate transcription. This NGFI-A-binding protein, NAB1, is a 570-aa nuclear protein that bears no obvious sequence homology to known proteins. NAB1 also represses Krox20 activity, but it does not influence Egr3 or NGFI-G, thus providing a mechanism for the differential regulation of this family of immediate-early transcription factors.

Submillisecond folding of monomeric lambda repressor.

The folding kinetics of a truncated form of the N-terminal domain of phage lambda repressor [lambda 6-85] has been investigated by using the technique of dynamic NMR. lambda 6-85 has been shown previously to fold in a purely two-state fashion. This allows the determination of folding and unfolding rates from simulation of the exchange-broadened aromatic resonances of Tyr-22. The folding kinetics were determined over a range of 1.35 to 3.14 M urea. The urea dependence of both folding and unfolding rate constants is exponential, suggesting that the rate-determining step is invariant at the urea concentrations studied. The folding and unfolding rates extrapolated to 0 M urea at 37 degrees C are 3600 +/- 400 s-1 and 27 +/- 6 s-1, respectively. The observed lambda 6-85 folding rate constant exceeds that of other fast-folding globular proteins by a factor of 14-54. The urea dependence of the folding and unfolding rate constants suggests that the transition state of the rate-determining step is considerably more exposed to solvent than previously studied protein-folding transition states. The surprising rapidity of lambda 6-85 folding and unfolding may be the consequence of its all-helical secondary structure. These kinetic results clearly demonstrate that all of the fundamental events of protein folding can occur on the submillisecond time scale.

Binding of purified multiple antibiotic-resistance repressor protein (MarR) to mar operator sequences.

Elevated expression of the marORAB multiple antibiotic-resistance operon enhances the resistance of Escherichia coli to various medically significant antibiotics. Transcription of the operon is repressed in vivo by the marR-encoded protein, MarR, and derepressed by salicylate and certain antibiotics. The possibility that repression results from MarR interacting with the marO operator-promoter region was studied in vitro using purified MarR and a DNA fragment containing marO. MarR formed at least two complexes with marO DNA, bound > 30-fold more tightly to it than to salmon sperm DNA, and protected two separate 21-bp sites within marO from digestion by DNase I. Site I abuts the downstream side of the putative -35 transcription-start signal and includes 4 bp of the -10 signal. Site II begins 13 bp downstream of site I, ending immediately before the first base pair of marR. Site II, approximately 80% homologous to site I, is not required for repression since a site II-deleted mutant (marO133) was repressed in trans by wild-type MarR. The absence of site II did not prevent MarR from complexing with the site I of marO133. Salicylate bound to MarR (Kd approximately 0.5 mM) and weakened the interaction of MarR with sites I and II. Thus, repression of the mar operon, which curbs the antibiotic resistance of E. coli, correlates with the formation of MarR-site I complexes. Salicylate appears to induce the mar operon by binding to MarR and inhibiting complex formation, whereas tetracycline and chloramphenicol, which neither bind MarR nor inhibit complex formation, must induce by an indirect mechanism.

In vivo estradiol-dependent dephosphorylation of the repressor MDBP-2-H1 correlates with the loss of in vitro preferential binding to methylated DNA.

We have previously shown that estradiol treatment of roosters resulted in a rapid loss of binding activity of the repressor MDBP-2-H1 (a member of the histone H1 family) to methylated DNA that was not due to a decrease in MDBP-2-H1 concentration. Here we demonstrate that MDBP-2-H1 from rooster liver nuclear extracts is a phosphoprotein. Phosphoamino acid analysis reveals that the phosphorylation occurs exclusively on serine residues. Two-dimensional gel electrophoresis and tryptic phosphopeptide analysis show that MDBP-2-H1 is phosphorylated at several sites. Treatment of roosters with estradiol triggers a dephosphorylation of at least two sites in the protein. Phosphatase treatment of purified rooster MDBP-2-H1 combined with gel mobility shift assay indicates that phosphorylation of MDBP-2-H1 is essential for the binding to methylated DNA and that the dephosphorylation can occur on the protein bound to methylated DNA causing its release from DNA. Thus, these results suggest that in vivo modification of the phosphorylation status of MDBP-2-H1 caused by estradiol treatment may be a key step for the down regulation of its binding to methylated DNA.

Repression by SSN6-TUP1 is directed by MIG1, a repressor/activator protein.

The SSN6-TUP1 protein complex represses transcription of diversely regulated genes in the yeast Saccharomyces cerevisiae. Here we present evidence that MIG1, a zinc-finger protein in the EGR1/Zif268 family, recruits SSN6-TUP1 to glucose-repressed promoters. DNA-bound LexA-MIG1 represses transcription of a target gene in glucose-grown cells, and repression requires SSN6 and TUP1. We also show that MIG1 and SSN6 fusion proteins interact in the two-hybrid system. Unexpectedly, we found that LexA-MIG1 activates transcription strongly in an ssn6 mutant and weakly in a tup1 mutant. Finally, LexA-MIG1 does not repress transcription in glucose-deprived cells, and MIG1 is differentially phosphorylated in response to glucose availability. We suggest a role for phosphorylation in regulating repression.

Nagorno-Karabakh and the arc of crises on Europe's borders. EPC Policy Brief, 3 February 2016

For more than two decades, Azerbaijan and Armenia have been locked in a stalemate over the Nagorno-Karabakh. The protracted conflict remains the biggest impediment to security, stability and prosperity in the South Caucasus. The EU has put itself on the sidelines of the conflict resolution process, allowing the Organization for Security and Co-operation in Europe (OSCE) Minsk Group (MG), of which Russia, France and the US are co-chairs, to take centre stage. In this Policy Brief, Amanda Paul and Dennis Sammut argue that the EU should play a more active role in the conflict resolution process, taking the lead with innovative initiatives and using its soft power skills and experience. A recent review of the European Neighbourhood Policy recognises that protracted conflicts continue to hamper development in the region. This new approach now needs to be given substance, before the conflict further escalates and becomes another crisis on Europe’s border the already burdened Union cannot cope with.

Chemistry and boron isotope ratios of Izu arc front volcanic rocks

The process of fluid release from the subducting slab beneath the Izu arc volcanic front (Izu VF) was examined by measuring B concentrations and B isotope ratios in the Neogene fallout tephra (ODP Site 782A). Both were measured by secondary ion mass spectrometry, in a subset of matrix glasses and glassy plagioclase-hosted melt inclusions selected from material previously analyzed for major and trace elements (glasses) and radiogenic isotopes (Sr, Nd, Pb; bulk tephra). These tephra glasses have high B abundances (~10-60 ppm) and heavy delta11B values (+4.5? to +12.0?), extending the previously reported range for Izu VF rocks (delta11B, +7.0? to +7.3?). The glasses show striking negative correlations of delta11B with large ion lithophile element (LILE)/Nb ratios. These correlations cannot be explained by mixing two separate slab fluids, originating from the subducting sediment and the subducting basaltic crust, respectively (model A). Two alternative models (models B and C) are proposed. Model B proposes that the inverse correlations are inherited from altered oceanic crust (AOC), which shows a systematic decrease of B and LILE with increasing depth (from basaltic layer 2A to layer 3), paralleled by an increase in delta11B (from ~ +1? to +10? to +24?). In this model, the contribution of sedimentary B is insignificant (<4% of B in the Izu VF rocks). Model C explains the correlation as a mixture of a low-delta11B (~ +1?) 'composite' slab fluid (a mixture of metasediment- and metabasalt-derived fluids) with a metasomatized mantle wedge containing elevated B (~1-2 ppm) and heavy delta11B (~ +14?). The mantle wedge was likely metasomatized by 11B-rich fluids beneath the outer forearc, and subsequently down dragged to arc front depths by the descending slab. Pb-B isotope systematics indicate that, at arc front depths, ~ 53% of the B in the Izu VF is derived from the wedge. This implies that the heavy delta11B values of Izu VF rocks are largely a result of fluid fractionation, and do not reflect variations in slab source provenance (i.e. subducting sediment vs. basaltic crust). Since the B content of the peridotite at the outer forearc (7-58 ppm B, mean 24 +/- 16 ppm) is much higher than beneath the arc front (~1-2 ppm B), the hydrated mantle wedge must have released a B-rich fluid on its downward path. This 'wedge flux' can explain (1) the across-arc decrease in B and delta11B (e.g. Izu, Kuriles), without requiring a progressive decrease in fluid flux from the subducting slab, and (2) the thermal structure of volcanic arcs, as reflected in the B and delta11B variations of volcanic arc rocks.

Age and chemical composition of dredged igneous rocks from the Vityaz Ridge, Pacific slope of the Kuril Island Arc

Original results of igneous rock studies are presented. The rocks were dredged during a marine expedition (cruise 37 of R/V Akademik M.A. Lavrent'ev in August-September, 2005) in the region of the submarine Vityaz Ridge and the Kuril Arc outer slope. Several age complexes (Late Cretaceous, Eocene, Late Oligocene, Miocene, and Pliocene-Pleistocene) are recognizable on the Vityaz Ridge. These complexes are characterized by a number of common geochemical features since all of them represent formations of island arc calc-alkali series. At the same time, they also have individual features reflecting different geodynamic settings. The outer slope of the Kuril Arc demonstrates submarine volcanism. Pliocene-Pleistocene volcanic rocks dredged here are similar to volcanites of the Kuril-Kamchatka Arc frontal zone.

Mission du Service géographique de l'Armée pour la mesure d'un arc de meridien équatorial en Amérique du Sud, sous le contrôl scientifique de l'Académie des sciences, 1899-1906.

Mode of access: Internet.

Joan of Arc, English mail coach, and Spanish military nun.

Mode of access: Internet.

La bienhaureuse Jeanne D'Arc, drama historique en quatre actes et douze tableaux.

Mode of access: Internet.