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Activation of the yeast GAL genes - localisation and interaction of transcriptional regulators

Reference	BB/C513477/1
Principal Investigator / Supervisor	Professor Richard Reece
Co-Investigators / Co-Supervisors
Institution	The University of Manchester
Department	Life Sciences
Funding type	Research
Value (£)	211,442
Status	Completed
Type	Research Grant
Start date	01/05/2005
End date	01/02/2009
Duration	45 months

Abstract

To survive and flourish, an organism must tightly regulate the expression of sets of genes. Certain gene products may only be needed in the cell for a brief and specific time, and the constitutive production of proteins can be deleterious to the cell. 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 (eg. 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 of the GAL genes of the yeast Saccharomyces cerevisiae. 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 at least 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. What is unclear, however, is how the interaction between Gal3p and Gal80p leads to the release of the transcriptional inhibition of Gal4p. Biochemical data suggests that a Gal4p-Gal80p-Gal3p complex forms in the presence of galatose and ATP that is transcriptionally competent. Other data, however, suggests that Gal3p is an exclusively cytoplasmic protein that the interaction between Gal3p and Gal80p may reduce the nuclear concentration of Gal80p hereby allowing Gal4p to recruit RNA polymerase II to the promoters of the GAL gene and transcription occurs. We will undertake a series of experiments to attempt to reconcile these apparently different proposed mechanisms of action. In pairs, the genes encoding the GAL regulatory proteins will be tagged (initially at their 3 prime ends) with the DNA sequence encoding either EYCP or ECFP (or alternative fluorescent proteins if required). Each tagged protein will be rigorously examined to ensure that it is expressed, produced and functions in an identical fashion to its wild-type counterpart. We will then use fluorescence resonance energy transfer (FRET) in living intact yeast cells to determine the cellular location of the interaction between each of these proteins. A combination of FRET and traditional fluorescence microscopy will be used to determine the cellular location of each of the GAL regulatory proteins and the location of the complexes in which they partake before, during, and after induction with galactose. In addition, the GAL regulatory complex proteins will be purified from yeast cells using TAP-tags. Combined, these data will generate a detailed picture of the fate and consequences of each of the GAL regulatory proteins during the process of transcriptional induction. Finally, FRET analysis will be performed on the GAL structural genes (Gal2p, Gal1p, Gal7p and Gal10p) to determine whether these cytoplasmic proteins form a metabolon that may assist in the channelling of metabolic intermediates during the enzymatic steps of the Leloir pathway. Taken together, the information generated through this project will shed new light on this important, but still poorly understood, regulatory system. The GAL system is often used as a text-book 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.

Summary

unavailable

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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