The problem of selective gene expression is of fundamental importance for all living cells. Cells have to respond to a great number of internal and external stimuli (or anti-stimuli) that range from metabolic signals to hormones to growth factors. The response often occurs at the level of gene transcription. Aberrant responses can lead to severe malfunction, disease, and cell death. This proposal focuses on the mechanism of action of a transcriptional activator of yeast that has several outstanding features. The regulator, a large DNA binding protein encoded by the LEU3 gene, acts on a number of RNA polymerase II-transcribed genes that are scattered throughout the genome. The Leu3 protein is a genetic switch that can act both as a repressor and as a metabolite-dependent activator of gene expression. The metabolite, alpha-isopropylmalate, is an intermediate in leucine biosynthesis. It is thought to serve as a signal, causing conformational changes that switch Leu3 from the repressing to the activating mode. Leu3's in vivo behavior can be faithfully reproduced in an in vitro transcription system, which opens new avenues of investigation. The following approaches are planned: (i) To analyze the structural and functional significance of the newly discovered phosphorylation of Leu3. (ii) To study the activation, alpha- isopropylmalate response, and repression functions of Leu3, which includes attempts at identifying the partner(s) within the transcription apparatus Leu3 might react with. (iii) To obtain information on the solution structure of the DNA binding domain of Leu3 and to study other aspects of the DNA binding function. (iv) To search for mutants of the LEU3 regulatory system in an attempt to identify additional structurally (and functionally) altered Leu3 molecules and partners of or substitutes for the Leu3 protein. Given the fact that Leu3 functions as activator not only in yeast, but also in mouse fibroblasts, it is reasonable to assume that knowledge gained from working with the Leu protein will increase our knowledge of eukaryotic transcriptional strategies in general.
