Functional characterization of transcriptional regulatory elements in the upstream region of the yeast GLK1 gene

The glucokinase gene GLK1 of the yeast Saccharomyces cerevisiae is transcri ptionally regulated in response to the carbon source of the growth medium. Northern-blot analysis shows that the GLK1 gene is expressed at a basal lev el in the presence of glucose, de-repressed more than 6-fold under conditio ns of sugar limitation and more than 25-fold under conditions of ethanol in duction, lacZ fusions of the GLK1 gene promoter were constructed and a dele tion analysis was performed in order to identify the cis-acting regulatory elements of the promoter that controls GLK1 gene expression. First, the exp ression seemed to be mediated mainly by one GCR1 and three stress-responsiv e element (STRE) activating elements. Secondly, an ethanol repression autor egulation (ERA)/twelve-fold TA repeat (TAB) repressor element was identifie d within the promoter region of the GLK1 gene. A specific and differential protein binding to the STRE was observed with extracts from de-repressed an d repressed cells. No differential binding to the GCR1 or ERA/TAB elements was observed with extracts from de-repressed and repressed cells, but, in b oth cases, the binding was competed for by an excess of the unlabelled GLK1 (GCR1) and GLK1(ERA) sequence. The transcription factors Msn2 and Msn4, whi ch bind to the GLK1 upstream region through the STRE, contribute to inducti ve activation. The transcription factor Gcr1, which binds through the GCR1 element, contributes to constitutive activation. In order to achieve the se vere glucose repression of GLK1, constitutive repressor factors acting thro ugh the ERA/TAB element must counteract constitutive activation generated b y Gcr1 binding to the GCR1 element. Full expression of the GLK1 gene is pro duced by inductive activation of three STRE when Msn2 and Msn4 proteins are translocated to the nucleus by covalent modification. The combinatorial ef fect of the entire region leads to the regulated transcription of GLK1, i.e ., silent in media with glucose and other preferred carbon sources, such as fructose or mannose, and increased levels of expression upon glucose deple tion.