Mediator is an intrinsic component of the basal RNA polymerase II machinery in vivo.

Mediator is a prominent multisubunit coactivator that functions as a bridge between gene-specific activators and the basal RNA polymerase (Pol) II initiation machinery. Here, we study the poorly documented role of Mediator in basal, or activator-independent, transcription in vivo. We show that Mediator is still present at the promoter when the Pol II machinery is recruited in the absence of an activator, in this case through a direct fusion between a basal transcription factor and a heterologous DNA binding protein bound to the promoter. Moreover, transcription resulting from activator-independent recruitment of the Pol II machinery is impaired by inactivation of the essential Mediator subunit Med17 due to the loss of Pol II from the promoter. Our results strongly support that Mediator is an integral component of the minimal machinery essential in vivo for stable Pol II association with the promoter.

Mediator, known as a coactivator, can act in transcription initiation in an activator-independent manner in vivo.

Mediator is an evolutionarily conserved complex best known for its role as a coactivator responsible for transducing regulatory signals from DNA-bound activators to the basal RNA polymerase II (Pol II) machinery that initiates transcription from promoters of protein-encoding genes. By exploiting our in vivo activator-independent transcription assay in Saccharomyces cerevisiae, in combination with new temperature sensitive (ts) mutants of Med14 N-terminal half exhibiting widespread transcriptional defects, and existing ts mutants of Kin28 and Med17, we show that, in the absence of activator: (i) Mediator can associate with a promoter as a form devoid of the Cyclin-dependent kinase 8 (CDK8) module, and this association remains regulated by Kin28; (ii) Mediator can stimulate the assembly of the entire Pol II initiation machinery. Although the literature emphasizes the role of the interaction between activators and Mediator, together our results support the view that Mediator is able to act through a dual mechanism in vivo, activator-dependent but also activator-independent, therefore not always as a coactivator.

Structurally conserved alpha-neurotoxin genes encode functionally diverse proteins in the venom of Naja sputatrix.

The structure and organization of the genes encoding the long-chain neurotoxins and four other isoforms of weak neurotoxins in the venom of Naja sputatrix are reported. The genes contained three exons interrupted by two introns, a structure similar to other members of the three-finger toxin family. The proteins encoded by these genes, however, show varied affinity towards nicotinic acetylcholine receptors. Phylogenetic analysis of these genes showed that the weak neurotoxin gene is confined to a distinct group. We also observe that specific mutations of the gene provide the diversity in function in these toxins while maintaining a common structural scaffold. This forms the first report where the molecular basis of evolution of postsynaptic neurotoxins from an ancestral gene can be demonstrated using the same species of snake.

Oligomerization of the SPP1 scaffolding protein.

Viral scaffolding proteins direct polymerization of major capsid protein subunits into icosahedral procapsid structures. The scaffolding protein of bacteriophage SPP1 was engineered with a C-terminal hexahistidine tag (gp11-His(6)) and purified. The protein is an alpha-helical-rich molecule with a very elongated shape as found for internal scaffolding proteins from other phages. It is a 3.3 S tetramer of 93.6 kDa at micromolar concentrations. Intersubunit cross-linking of these tetramers generated preferentially covalently bound dimers, revealing that gp11-His(6) is structurally a dimer of dimers. Incubation at temperatures above 37 degrees C correlated with a reduction of its alpha-helical content and a less effective intersubunit cross-linking. Complete loss of secondary structure was observed at temperatures above 60 degrees C. Refolding of gp11-His(6) thermally denatured at 65 degrees C led to reacquisition of the protein native ellipticity spectrum but the resulting population of molecules was heterogeneous. Its hydrodynamic behavior was compatible with a mix of 3.3 S elongated tetramers (approximately 90%) and a smaller fraction of 2.4 S dimers (approximately 10%). This population of gp11-His(6) was competent to direct polymerization of the SPP1 major capsid protein gp13 into procapsid-like structures in a newly developed assembly assay in vitro. Although native tetramers were active in assembly, refolded gp11-His(6) showed enhanced binding to gp13 revealing a more active species for interaction with the major capsid protein than native gp11-His(6).

Systematic deletion analysis of fission yeast protein kinases.

Eukaryotic protein kinases are key molecules mediating signal transduction that play a pivotal role in the regulation of various biological processes, including cell cycle progression, cellular morphogenesis, development, and cellular response to environmental changes. A total of 106 eukaryotic protein kinase catalytic-domain-containing proteins have been found in the entire fission yeast genome, 44% (or 64%) of which possess orthologues (or nearest homologues) in humans, based on sequence similarity within catalytic domains. Systematic deletion analysis of all putative protein kinase-encoding genes have revealed that 17 out of 106 were essential for viability, including three previously uncharacterized putative protein kinases. Although the remaining 89 protein kinase mutants were able to form colonies under optimal growth conditions, 46% of the mutants exhibited hypersensitivity to at least 1 of the 17 different stress factors tested. Phenotypic assessment of these mutants allowed us to arrange kinases into functional groups. Based on the results of this assay, we propose also the existence of four major signaling pathways that are involved in the response to 17 stresses tested. Microarray analysis demonstrated a significant correlation between the expression signature and growth phenotype of kinase mutants tested. Our complete microarray data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/kinome.

A synthetic weak neurotoxin binds with low affinity to Torpedo and chicken alpha7 nicotinic acetylcholine receptors.

Weak neurotoxins from snake venom are small proteins with five disulfide bonds, which have been shown to be poor binders of nicotinic acetylcholine receptors. We report on the cloning and sequencing of four cDNAs encoding weak neurotoxins from Naja sputatrix venom glands. The protein encoded by one of them, Wntx-5, has been synthesized by solid-phase synthesis and characterized. The physicochemical properties of the synthetic toxin (sWntx-5) agree with those anticipated for the natural toxin. We show that this toxin interacts with relatively low affinity (K(d) = 180 nm) with the muscular-type acetylcholine receptor of the electric organ of T. marmorata, and with an even weaker affinity (90 microm) with the neuronal alpha7 receptor of chicken. Electrophysiological recordings using isolated mouse hemidiaphragm and frog cutaneous pectoris nerve-muscle preparations revealed no blocking activity of sWntx-5 at microm concentrations. Our data confirm previous observations that natural weak neurotoxins from cobras have poor affinity for nicotinic acetylcholine receptors.