Kinetic competition between elongation rate and binding of NELF controls promoter-proximal pausing.

Pausing of RNA polymerase II (Pol II) 20-60 bp downstream of transcription start sites is a major checkpoint during transcription in animal cells. Mechanisms that control pausing are largely unknown. We developed permanganate-ChIP-seq to evaluate the state of Pol II at promoters throughout the Drosophila genome, and a biochemical system that reconstitutes promoter-proximal pausing to define pausing mechanisms. Stable open complexes of Pol II are largely absent from the transcription start sites of most mRNA genes but are present at snRNA genes and the highly transcribed heat shock genes following their induction. The location of the pause is influenced by the timing between when NELF loads onto Pol II and how fast Pol II escapes the promoter region. Our biochemical analysis reveals that the sequence-specific transcription factor, GAF, orchestrates efficient pausing by recruiting NELF to promoters before transcription initiation and by assisting in loading NELF onto Pol II after initiation.

LaOx modified MnOx loaded biomass activated carbon and its enhanced performance for simultaneous abatement of NO and Hg0.

A battery of agricultural straw derived biomass activated carbons supported LaOx modified MnOx (LaMn/BACs) was prepared by a facile impregnation method and then tested for simultaneous abatement of NO and Hg0. 15%LaMn/BAC manifested excellent removal efficiency of Hg0 (100%) and NO (86.7%) at 180 °C, which also exhibited splendid resistance to SO2 and H2O. The interaction between Hg0 removal and NO removal was explored; thereinto, Hg0 removal had no influence on NO removal, while NO removal preponderated over Hg0 removal. The inhibitory effect of NH3 was greater than the accelerative effect of NO and O2 on Hg0 removal. The physicochemical characterization of related samples was characterized by SEM, XRD, BET, H2-TPR, NH3-TPD, and XPS. After incorporating suitable LaOx into 15%Mn/BAC, the synergistic effect between LaOx and MnOx contributed to the improvement of BET surface area and total pore volume, the promotion of redox ability, surface active oxygen species, and acid sites, inhibiting the crystallization of MnOx. 15%LaMn/BAC has the best catalytic oxidation activity at low temperature. That might be answerable for superior performance and preferable tolerance to SO2 and H2O. The results indicated that 15%LaMn/BAC was a promising catalyst for simultaneous abatement of Hg0 and NO at low temperature.

GAGA factor and the TFIID complex collaborate in generating an open chromatin structure at the Drosophila melanogaster hsp26 promoter.

The upstream regulatory region of the Drosophila melanogaster hsp26 gene includes two DNase I-hypersensitive sites (DH sites) that encompass the critical heat shock elements. This chromatin structure is required for heat shock-inducible expression and depends on two (CT)n*(GA)n elements bound by GAGA factor. To determine whether GAGA factor alone is sufficient to drive formation of the DH sites, we have created flies with an hsp26/lacZ transgene wherein the entire DNA segment known to interact with the TFIID complex has been replaced by a random sequence. The replacement results in a loss of heat shock-inducible hsp26 expression and drastically diminishes nuclease accessibility in the chromatin of the regulatory region. Chromatin immunoprecipitation experiments show that the decrease in TFIID binding does not reduce GAGA factor binding. In contrast, the loss of GAGA factor binding resulting from (CT)n mutations decreases TFIID binding. These data suggest that both GAGA factor and TFIID are necessary for formation of the appropriate chromatin structure at the hsp26 promoter and predict a regulatory mechanism in which GAGA factor binding precedes and contributes to the recruitment of TFIID.

DNA-binding domain of GCN4 induces bending of both the ATF/CREB and AP-1 binding sites of DNA.

The interaction of proteins with DNA results, in some cases, in DNA bending, and this might have functional importance. However, when the protein-induced bending of DNA is small, its measurement presents a problem. It is shown that the fluorescence resonance energy transfer between fluorophores placed on the ends of the specially designed U-shaped DNA, which contains the DNA-binding sites at its central part, can be successfully used for this purpose. The lever effect of the arms of such U-shaped DNA ensures that the distance between the fluorophores is very sensitive to bending of the central part. Using this technique, it was shown that (i) the AP-1 and ATF/CREB binding sites of GCN4 transcription factor are pre-bent to the same extent (approximately 12 degrees toward the major groove) and (ii) binding of the GCN4 DNA-binding domain (GCN4-bZIP) results in additional bending of both these target sites but to a greater extent at the ATF/CREB site. In total, in the complex with GCN4-bZIP, the ATF/CREB site is bent by (25 +/- 2) degrees and the AP-1 site by (20 +/- 2) degrees toward the minor groove.

Thermodynamic signature of GCN4-bZIP binding to DNA indicates the role of water in discriminating between the AP-1 and ATF/CREB sites.

The energetic basis of GCN4-bZIP complexes with the AP-1 and ATF/CREB sites was investigated by optical methods and scanning and isothermal titration microcalorimetry. The dissociation constant of the bZIP dimer was found to be significantly higher than that of its isolated leucine zipper domain: at 20 degrees C it is 1.45microM and increases with temperature. To avoid complications from dissociation of this dimer, DNA binding experiments were carried out using an SS crosslinked version of the bZIP. The thermodynamic characteristics of the bZIP/DNA association measured at different temperatures and salt concentrations were corrected for the contribution of refolding the basic segment upon binding, determined from the scanning calorimetric experiments. Fluorescence anisotropy titration experiments showed that the association constants of the bZIP at 20 degrees C with the AP-1 and ATF/CREB binding sites do not differ much, being 1.5nM and 6.4nM, corresponding to Gibbs energies of -49kJmol(-1) and -46kJmol(-1), respectively. Almost half of the Gibbs energy is attributable to the electrostatic component, resulting from the entropic effect of counterion release upon DNA association with the bZIP and is identical for both sites. In contrast to the Gibbs energies, the enthalpies of association of the fully folded bZIP with the AP-1 and ATF/CREB sites, and correspondingly the entropies of association, are very different. bZIP binding to the AP-1 site is characterized by a substantially larger negative enthalpy and non-electrostatic entropy than to the ATF/CREB site, implying that the AP-1 complex incorporates significantly more water molecules than the ATF/CREB complex.

Optimization of CD4/gp120 inhibitors by thermodynamic-guided alanine-scanning mutagenesis.

As protein/protein interactions usually trigger signalling processes, inhibitors of those interactions must preclude protein binding without eliciting the signalling process themselves. To accomplish those goals, small molecules need to target those protein residues that contribute the most to binding (binding hotspots) without disturbing those residues that initiate signalling processes (allosteric hotspots). The availability of a blueprint identifying binding and allosteric hotspots will significantly aid inhibitor design and optimization. In this study, we show that in some situations the blueprint can be constructed by combining the standard technique of alanine-scanning mutagenesis with isothermal titration calorimetry (ITC). We demonstrate the approach by developing the combined binding and allosteric hotspots blueprint for CD4/gp120, the initial interaction leading to HIV-1 cell infection. A major finding of these studies is that not all binding hotspots are allosteric hotspots opening the possibility for the rational design of inhibitors and antagonist or agonist modulators.

Forces driving the binding of homeodomains to DNA.

Homeodomains are helix-turn-helix type DNA-binding domains that exhibit sequence-specific DNA binding by insertion of their "recognition" alpha helices into the major groove and a short N-terminal arm into the adjacent minor groove without inducing substantial distortion of the DNA. The stability and DNA binding of four representatives of this family, MATalpha2, engrailed, Antennapedia, and NK-2, and truncated forms of the last two lacking their N-terminal arms have been studied by a combination of optical and microcalorimetric methods at different temperatures and salt concentrations. It was found that the stability of the free homeodomains in solution is rather low and, surprisingly, is reduced by the presence of the N-terminal arm for the Antennapedia and NK-2 domains. Their stabilities depend significantly upon the presence of salt: strongly for NaCl but less so for NaF, demonstrating specific interactions with chloride ions. The enthalpies of association of the homeodomains with their cognate DNAs are negative, at 20 degrees C varying only between -12 and -26 kJ/mol for the intact homeodomains, and the entropies of association are positive; i.e., DNA binding is both enthalpy- and entropy-driven. Analysis of the salt dependence of the association constants showed that the electrostatic component of the Gibbs energy of association resulting from the entropy of mixing of released ions dominates the binding, being about twice the magnitude of the nonelectrostatic component that results from dehydration of the protein/DNA interface, van der Waals interactions, and hydrogen bonding. A comparison of the effects of NaCl/KCl with NaF showed that homeodomain binding results in a release not only of cations from the DNA phosphates but also of chloride ions specifically associated with the proteins. The binding of the basic N-terminal arms in the minor groove is entirely enthalpic with a negative heat capacity effect, i.e., is due to sequence-specific formation of hydrogen bonds and hydrophobic interactions rather than electrostatic contacts with the DNA phosphates.