No abstract

A central issue in eukaryotic transcriptional regulation is the mechanism by which promoter-specific transcription factors (activators) stimulate transcription. Two lines of evidence indicate that the general transcription factor TFIIB is a pivotal component in the mechanism by which an acidic activator functions. First, during assembly of the preinitiation complex TFIIB binding is a rate-limiting step enhanced by an acidic activator. Second, the TFIIB activity in a HeLa cell nuclear extract is specifically retained on a column containing an acidic activating region. But because our previous study monitored only TFIIB activity, it remains possible that the interaction between TFIIB and the acidic activating region is mediated through additional proteins, for example, those designated as adaptors, coactivators or mediators. A complementary clone encoding TFIIB has recently been isolated and shown to encode a polypeptide of relative molecular mass 35,000. Here we report that TFIIB expressed in and purified from Escherichia coli (recombinant TFIIB) binds directly to the potent acidic activating region of the herpes simplex virus-1 VP16 protein.

Three-dimensional renal tissues that emulate the cellular composition, geometry, and function of native kidney tissue would enable fundamental studies of filtration and reabsorption. Here, we have created 3D vascularized proximal tubule models composed of adjacent conduits that are lined with confluent epithelium and endothelium, embedded in a permeable ECM, and independently addressed using a closed-loop perfusion system to investigate renal reabsorption. Our 3D kidney tissue allows for coculture of proximal tubule epithelium and vascular endothelium that exhibits active reabsorption via tubular–vascular exchange of solutes akin to native kidney tissue. Using this model, both albumin uptake and glucose reabsorption are quantified as a function of time. Epithelium–endothelium cross-talk is further studied by exposing proximal tubule cells to hyperglycemic conditions and monitoring endothelial cell dysfunction. This diseased state can be rescued by administering a glucose transport inhibitor. Our 3D kidney tissue provides a platform for in vitro studies of kidney function, disease modeling, and pharmacology.

In prokaryotes and eukaryotes many gene activators work synergistically. For example, two dimers of lambda repressor interact to promote binding of these proteins to DNA, a reaction that is crucial at the repressor concentrations found in lysogens. In this case one of the bound dimers activates transcription, evidently by touching RNA polymerase. In another example, the yeast transcriptional activator GAL4, which can stimulate transcription in many eukaryotes, binds to multiple sites on DNA to activate transcription synergistically; the presence of two such sites can elicit a level of transcription more than twice that found with a single site. In this paper we show that synergistic activation by each of several GAL4 derivatives involves a mechanism different from that illustrated by the lambda repressor: multiple activator molecules can work synergistically under conditions in which their binding sites on DNA are saturated. The accompanying paper shows that under similar conditions of activator excess, GAL4 derivatives work synergistically with a heterologous mammalian gene activator. These results support the idea that eukaryotic activators can cooperate not by directly interacting but by simultaneously touching some component(s) of the transcriptional machinery.