Abstract
Mechanisms involved in transcriptional regulation of the osmotically controlled GPD1 gene in Saccharomyces cerevisiae were investigated by promoter analysis. The GPD1 gene encodes NAD(+)-dependent glycerol-3-phosphate dehydrogenase, a key enzyme in the production of the compatible solute glycerol. By analysis of promoter deletions, we identified a region at nucleotides -478 to -324, in relation to start of translation, to be of great importance for both basal activity and osmotic induction of GPD1. Electrophoretic mobility shift and DNase I footprint analyses demonstrated protein binding to parts of this region that contain three consensus sequences for Rap1p (repressor activator protein 1)-binding sites. Actual binding of Rap1p to this region was confirmed by demonstrating enhanced electrophoretic mobility of the protein-DNA complex with extracts containing an N-terminally truncated version of Rap1p. The detected Rap1p-DNA interactions were not affected by changes in the osmolarity of the growth medium. Specific inactivation of the Rap1p-binding sites by a C-to-A point mutation in the core of the consensus showed that this factor is a major determinant of GPD1 expression since mutations in all three putative binding sites for Rap1p strongly hampered osmotic induction and drastically lowered basal activity. We also show that the Rap1p-binding sites appear functionally distinct; the most distal site (core of the consensus at position -386) exhibited the highest affinity for Rap1p and was strictly required for low salt induction (< or =0.6 m NaCl), but not for the response at higher salinities (> or =0.8 m NaCl). This indicates tha different molecular mechanisms might be operational for low and high salt responses of the GPD1 promoter.
Highlights
Stress-activated signaling pathways in eukaryotic organisms are presently attracting much interest
To identify the element(s) of the GPD1 promoter responsible for the osmotically controlled transcriptional activation, a series of 5Ј-promoter deletions were constructed starting at nucleotide Ϫ687 in relation to the start of translation
Complex Regulation of the GPD1 Promoter—Insights into the mechanisms involved in the control of the GPD1 promoter will provide a major key to our general understanding of hyperosmotic stress-induced gene regulation
Summary
Stress-activated signaling pathways in eukaryotic organisms are presently attracting much interest. More recent work identified several upstream components of two distinct branches of the pathway affecting Hog1p phosphorylation [5,6,7,8] Another signaling pathway of general importance in modulating various cellular activities, i.e. the cAMP-dependent protein kinase A pathway, influences the expression of some of the stress-regulated genes [1, 9] by opposing the effects from signaling in the HOG pathway [10]. We present evidence for the binding of Rap1p to neighboring binding sites that appear involved in mediating regulatory effects upon dehydration via different mechanisms, depending on the magnitude of stress This is the first time that this multifunctional transcription factor is being implicated in the osmostress response. It is hypothesized, based on the importance of Rap1p in the Gcr1p-mediated induction of glycolytic genes, that Rap1p might interact directly or indirectly with Hot1p and/or Msn1p and thereby facilitate their binding and subsequent activation of the GPD1 promoter
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