Structural and functional characterization of the yeast GAL genetic switch

The genome of an organism is complex. It contains within its DNA sequence all the information required to define not only cell type, but also the ability to respond to a variety of external conditions and signals. For example, the genome of the simple eukaryotic yeast Saccharomyces cerevisiae contains 12 million base-pairs of DNA split into 16 separate chromosomes and comprises some 6,000 different genes. The protein products of these genes rarely act individually, and whole pathways are often regulated in response to a particular signal. Indeed, the complex processes of the cell depend on differential expression of sets of genes either in particular cell types (e.g., when cell differentiation takes place within an embryo), at a particular time (e.g., when a microbe produces an antibiotic late in its growth cycle), or under certain environmental conditions (e.g., changes in the metabolic flux within a cell depending upon metabolite availability). Thus within cells there are programmes whereby sets of genes can be co-ordinately switched on or off. Efficient switches, must be able to operate in two directions: on to off and off to on. Genetic switches must operate in this way also, for example allowing appropriate gene expression when the certain nutrients are available, and turning gene expression off when nutrients are limiting or are unavailable. Insights into these processes can be gained by studying the relatively simple genetic switches of yeast (although the mechanistic details have turned out to be frighteningly complex!). Yeasts share a large number of similarities with human and other animal cells. The process of reading the information contained within a gene (transcription) is very similar between yeast and human cells. Indeed, many human transcription factors are able to functionally complement their yeast homologues. Yeast cells regulate many sets of genes in response to external signals. For example, yeasts grown on a variety of carbon sources do not express the enzymes for the degradation of galactose. However, when galactose is the sole carbon source available, the cell rapidly expresses large amounts of the galactose degradation enzymes. The galactose genetic switch involves at least three players; an activator (responsible for turning the genes on), a repressor (inhibits the activator), and an inducer that modulates the interaction of the activator and the repressor. The interplay between these three protein components is mediated by two small molecules / a sugar and a source of energy (galactose and ATP). The galactose switch (GAL switch) has been studied for many years using a combination of classical yeast genetic techniques and biochemical approaches. These methods have, however, generated conflicting results concerning the mechanism of gene activation in response to galactose. The work proposed here will resolve these conflicts by solving the three dimensional structures of each of the GAL regulatory proteins (both alone and in complex with each other). Combined, these data will elucidate the mechanism of action of this important genetic switch.

To survive and flourish an organism must tightly regulate the expression of sets of genes. To ensure proper timing and levels of gene expression, elaborate, and often overlapping, mechanisms exist within cells to detect changes in internal and external environmental conditions (e.g., levels of metabolites, the presence of a hormone, etc.) and convert the detection of a signal into a transcriptional response so that the produced proteins can mount a response to that particular signal. In eukaryotes, one of the most intensively studied transcriptional control system is that if the yeast GAL genes. This set of genes are only expressed when the cells are grown on the sugar galactose as the sole source of carbon. The expression of the GAL genes is regulated by three proteins / a transcriptional activator (Gal4p), a repressor (Gal80p) and an inducer (Gal3p). A physical association between Gal4p and Gal80p inhibits the transcriptional activation function of Gal4p. Induction of gene expression occurs when galactose and ATP bind to Gal3p and this protein-metabolite complex interacts with Gal80p. We have recently solved the structure of Gal80p at 2.1Å resolution, and have obtained crystals of Gal80p in complex with a peptide representing the acidic activation domain of Gal4p. Here, we will continue our high-resolution structural analysis of the GAL regulatory proteins to determine the precise chemical environments that exists between the three proteins. Taken together, the information generated through this project will shed new light on this important, but still comparatively poorly understood, regulatory system. The GAL system is often used as a textbook example of the control of gene expression in eukaryotes. It is time, therefore, to elucidate its mechanism more fully so that a clear and unambiguous picture of its method of regulation may be drawn.