Modulation of promoter occupancy by cooperative DNA binding and activation-domain function is a major determinant of transcriptional regulation by activators in vivo.

Binding of transcriptional activators to a promoter is a prerequisite process in transcriptional activation. It is well established that the efficiency of activator binding to a promoter is determined by the affinity of direct interactions between the DNA-binding domain of an activator and its specific target sequences. However, I describe here that activator binding to a promoter is augmented in vivo by the effects of two other determinants that have not been generally appreciated: (i) the number of activator binding sites present in a promoter and (ii) the potency of activation domains of activators. Multiple sites within a promoter can cooperatively recruit cognate factors regardless of whether they contain an effective activation domain. This cooperativity can result in the synergistic activation of transcription. The second effect is the enhancement of activator binding to a promoter by the presence of activation domains. In this case, activation domains are not simply tethered to the promoter by the DNA-binding domain but instead assist the DNA-binding domain being tethered onto the promoter. This effect of activation domains on DNA binding is instrumental in determining how potent activators can induce steep transcriptional increases at low concentrations.

Cooperative interactions among afferents govern the induction of homosynaptic long-term depression in the hippocampus.

Prolonged periods of low-frequency stimulation have been shown to produce a robust, long-term synaptic depression (LTD) in both hippocampus and visual cortex. In the present study we have examined the extent to which interactions among afferents govern the induction of homosynaptic LTD in young-adult rats in hippocampal region CA1 in vitro. Field excitatory postsynaptic potentials were assessed before and after conditioning stimulation consisting of two 10-min trains of low-frequency stimulation (LFS; 1 Hz) of the Schaffer collateral/commissural pathway. LFS at an intensity producing a 0.5-mV response did not produce significant synaptic depression. However, LFS administered at a higher intensity resulted in significant input-specific LTD of a 0.5-mV test response. Picrotoxin, which also facilitates depolarization of CA1 neurons, significantly enhanced the magnitude of LTD after LFS at 0.5 mV. In addition, LFS at 0.5 mV in normal perfusion medium (no picrotoxin) produced only small changes in synaptic efficacy when either of two converging pathways was conditioned separately but produced a robust LTD when both pathways were conditioned simultaneously. This cooperative LTD was reversibly blocked by prior administration of 100 microM DL-aminophosphonovaleric acid but not by 20 microM nimodipine. Taken together, these results suggest that cooperative interactions among afferents contribute to voltage-dependent processes underlying the induction of homosynaptic LTD.

The activation domain of GAL4 protein mediates cooperative promoter binding with general transcription factors in vivo.

Most proteins that activate RNA polymerase II-mediated transcription in eukaryotic cells contain sequence-specific DNA-binding domains and "activation" regions. The latter bind general transcription factors and/or coactivators and are required for high-level transcription. Their function in vivo is unknown. Since several activation domains bind the TATA-binding protein (TBP), TBP-associated factors, or other general factors in vitro, one role of the activation domain may be to facilitate promoter occupancy by supporting cooperative binding of the activator and general transcription factors. Using the GAL4 system of yeast, we have tested this model in vivo. It is demonstrated that the presence of a TATA box (the TBP binding site) facilitates binding of GAL4 protein to low- and moderate-affinity sites and that the activation domain modulates these effects. These results support the cooperative binding model for activation domain function in vivo.

A cooperative model for receptor recognition and cell adhesion: evidence from the molecular packing in the 1.6-A crystal structure of the pheromone Er-1 from the ciliated protozoan Euplotes raikovi.

The crystal structure of the pheromone Er-1 from the unicellular eukaryotic organism Euplotes raikovi was determined at 1.6 A resolution and refined to a crystallographic R factor of 19.9%. In the tightly packed crystal, two extensive intermolecular helix-helix interactions arrange the Er-1 molecules into layers. Since the putative receptor of the pheromone is a membrane-bound protein, whose extracellular C-terminal domain is identical in amino acid sequence to the soluble pheromone, the interactions found in the crystal may mimic the pheromone-receptor interactions as they occur on a cell surface. Based on this, we propose a model for the interaction between soluble pheromone molecules and their receptors. In this model, strong pheromone-receptor binding emerges as a consequence of the cooperative utilization of several weak interactions. The model offers an explanation for the results of binding studies and may also explain the adhesion between cells that occurs during mating.

DNA loops induced by cooperative binding of transcriptional activator proteins and preinitiation complexes.

DNA conformational changes are essential for the assembly of multiprotein complexes that contact several DNA sequence elements. An approach based on atomic force microscopy was chosen to visualize specific protein-DNA interactions occurring on eukaryotic class II nuclear gene promoters. Here we report that binding of the transcription regulatory protein Jun to linearized plasmid DNA containing the consensus AP-1 binding site upstream of a class II gene promoter leads to bending of the DNA template. This binding of Jun was found to be essential for the formation of preinitiation complexes (PICs). The cooperative binding of Jun and PIC led to looping of DNA at the protein binding sites. These loops were not seen in the absence of either PICs, Jun, or the AP-1 binding site, suggesting a direct interaction between DNA-bound Jun homodimers and proteins bound to the core promoter. This direct visualization of functional transcriptional complexes confirms the theoretical predictions for the mode of gene regulation by trans-activating proteins.

Cooperative transcriptional activity of Jun and Stat3 beta, a short form of Stat3.

To identify proteins that regulate the transcriptional activity of c-Jun, we have used the yeast two-hybrid screen to detect mammalian polypeptides that might interact functionally with the N-terminal segment of c-Jun, a known regulatory region. Among the proteins identified is a short form of Stat3 (designated Stat3 beta). Stat3 beta is missing the 55 C-terminal amino acid residues of the long form (Stat3 alpha) and has 7 additional amino acid residues at its C terminus. In the absence of added cytokines, expression of Stat3 beta (but not Stat3 alpha) in transfected cells activated a promoter containing the interleukin 6 responsive element of the rat alpha 2-macroglobulin gene; coexpression of Stat3 beta and c-Jun led to enhanced cooperative activation of the promoter. Nuclear extracts of cells transfected with a Stat3 beta expression plasmid formed a complex with an oligonucleotide containing a Stat3 binding site, whereas extracts of cells transfected with a Stat3 alpha plasmid did not. We conclude that there is a short form of Stat3 (Stat3 beta), that Stat3 beta is transcriptionally active under conditions where Stat3 alpha is not, and that Stat3 beta and c-Jun are capable of cooperative activation of certain promoters.

The hexapeptide LFPWMR in Hoxb-8 is required for cooperative DNA binding with Pbx1 and Pbx2 proteins.

The Hox gene products are DNA-binding proteins, containing a homeodomain, which function as a class of master control proteins establishing the body plan in organisms as diverse as Drosophila and vertebrates. Hox proteins have recently been shown to bind cooperatively to DNA with another class of homeodomain proteins that include extradenticle, Pbx1, and Pbx2. Hox gene products contain a highly conserved hexapeptide connected by a linker of variable length to the homeodomain. We show that the hexapeptide and the linker region are required for cooperativity with Pbx1 and Pbx2 proteins. Many of the conserved residues present in the Hoxb-8 hexapeptide are required to modulate the DNA binding of the Pbx proteins. Position of the hexapeptide relative to the homeodomain is important. Although deletions of two and four residues of the linker peptide still show cooperative DNA binding, removal of all six linker residues strongly reduces cooperativity. In addition, an insertion of 10 residues within the linker peptide significantly lowers cooperative DNA binding. These results show that the hexapeptide and the position of the hexapeptide relative to the homeodomain are important determinants to allow cooperative DNA binding involving Hox and Pbx gene products.

A variant of lambda repressor with an altered pattern of cooperative binding to DNA sites.

The bacteriophage lambda repressor binds cooperatively to pairs of adjacent sites in the lambda chromosome, one repressor dimer binding to each site. The repressor's amino domain (that which mediates DNA binding) is connected to its carboxyl domain (that which mediates dimerization and the interaction between dimers) by a protease-sensitive linker region. We have generated a variant lambda repressor that lacks this linker region. We show that dimers of the variant protein are deficient in cooperative binding to sites at certain, but not all, distances. The linker region thus extends the range over which carboxyl domains of DNA-bound dimers can interact. In particular, the linker is required for cooperative binding to a pair of sites as found in the lambda chromosome, and thus is essential for the repressor's physiological function.

Folding of a nascent polypeptide chain in vitro: cooperative formation of structure in a protein module.

We have prepared a family of peptide fragments of the 64-residue chymotrypsin inhibitor 2, corresponding to its progressive elongation from the N terminus. The growing polypeptide chain has little tendency to form stable structure until it is largely synthesized, and what structures are formed are nonnative and lack, in particular, the native secondary structural elements of alpha-helix and beta-sheet. These elements then develop as sufficient tertiary interactions are made in the nearly full-length chain. The growth of structure in the small module is highly cooperative and does not result from the hierarchical accretion of substructures.

Utilization of ADR Concepts to Bridge the Gap Between the Extractive Industry and Environmental Interests: An Anticipatory Cooperative Effort (ACE)

The extractive industry, more than any other sector of the economy, often finds itself mired in conflicts with various environmental and community interests. As traditional legal avenues of resolution gave way to the collaborative ideas of alternative dispute resolution, the outcomes, especially the relational outcomes, were less than desirable. This capstone project proposes that an Anticipatory Cooperative Effort (ACE) can help to bridge the gap between industry and environmental interests by encouraging a pro-active and pre-emptive engagement. The point of the ACE concept is not that it defines a new set of principles so much as it repositions where established ADR principles are entertained.

An Acyl-NHC Osmium Cooperative System: Coordination of Small Molecules and Heterolytic B–H and O–H Bond Activation

The hexahydride complex OsH6(PiPr3)2 (1) activates the C–OMe bond of 1-(2-methoxy-2-oxoethyl)-3-methylimidazolium chloride (2), in addition to promoting the direct metalation of the imidazolium group, to afford a five-coordinate OsCl(acyl-NHC)(PiPr3)2 (3) compound. The latter coordinates carbon monoxide, oxygen, and molecular hydrogen to give the corresponding carbonyl (4), dioxygen (5), and dihydrogen (6) derivatives. Complex 3 also promotes the heterolytic bond activation of pinacolborane (HBpin), using the acyl oxygen atom as a pendant Lewis base. The hydride ligand and the Bpin substituent of the Fischer-type carbene of the resulting complex 7 activate the O–H bond of alcohols and water. As a consequence, complex 3 is a metal ligand cooperating catalyst for the generation of molecular hydrogen, by means of both the alcoholysis and hydrolysis of pinacolborane, via the intermediates 7 and 6.

How can directories of open access repositories improve the reuse of learning objects in building engineering?

This paper studies the use of directories of open access repositories worldwide (DOARW) to search Spanish repositories containing learning objects in the field of building engineering (BE). Results show that DOARW are powerful tools, but deficiencies (indicated in this study) have to be solved in order to obtain more accurate searches, and to facilitate repository-finding for potential users who are seeking learning objects (LOs) for reuse. Aiming to contribute to the promotion of the reuse of Spanish LOs, this study exposes to the academic community all existing Spanish repositories with LOs, and in particular, the repositories that contain LOs in the field of BE. This paper also studies the critical mass of available content (LOs) in the field of BE in Spain. It has been found to be low.

Exploring the use of cooperative activities in a language subject to develop students’ social competences at the University

Cooperative learning has been successfully implemented in the last 60 years for teaching at different educational levels including the Higher Studies due to its solid theoretical foundation, the principles it proposes and its practical applications. The purpose of this article is to offer a proposal for some cooperative activities that allow students to work in small groups in a language subject in order to learn not only contents but also putting into practice what they learn, i.e., they learn by being active. This article discusses how the said activities make it possible for students to work with the main principles of cooperative learning, i.e.: positive interdependence, face-to-face interaction, individual and group accountability, interpersonal and small-group skills and group processing. Moreover, this research will also point out that the proposed activities allow students to acquire some of the social competences required in the labour market such as leadership, conflict solving and cooperation.

How to Use Cooperative Learning for Assessing Students’ Emotional Competences: A Practical Example at the Tertiary Level

Cooperative learning allows students acquisition of competences that are essential for the labour market such as leadership, critical thinking, communication, and so on. For this reason, different cooperative activities were designed in a language subject in English Studies so that students could work in groups and acquire those competences. This article describes some such activities and the emotional competences that students acquire with them. Moreover, a survey was conducted in order to establish students’ opinions about the main competences they acquired with the activities designed and their opinion about a cooperative methodology. Students’ answers were positive and they were aware of what they had learned.