The expression of B-galactosidase from each of these promoter deletion constructs under minus-galactose, plus-galactose, and plus galactose glucose, are show. From these data we can deduce the location of cis-acting regulatory sequences for the Gall gene Deletions 7 and 8 do not express the reporter under any conditions because the deletions have removed some of the tAta sequence that is required for assembly of the basal transcription machinery Deletions 1 and 2 eliminate the ability of galactose to increase expression from the Gall promoter, and since expression is not induced there is nothing for glucose to repress. It turns out that the 75bp sequence between -310 and -385 is the dna binding site for gal4 and this kind of region is generally called a UAs (upstream activation sequence) and in this case UASGAL. We will come back to thinking about Gal4 binding to the UAs recognition sequence later. Deletions 3, 5 and 6 have no effect on the ability of galactose to induce expression because the UAs remains intact. Note that shortening the distance between the UAs and the tata region is not detrimental to induction. Indeed increasing the distance by inserting extra dna between the UAs and the tata sequence also has little effect on inducibility. This has led to the idea that UAS sequences can work at long distances (1,000-10,000 bp) away from the tata sequence and the transcription start sites. (In mammalian cells regions containing binding sites for transcriptional activators are called enhancers we will come to these in a later lecture) Deletion 4 turns out to reveal information about glucose repression For this construct, while galactose induces expression glucose is unable to repress that expression. the deleted region defines the position of a sequence element needed for glucose repression and a sequence element that behaves this way (i.e. are required for repression) is generally called a URS (upstream repressor sequence), and in this case URSGAL No/low Glucose High Glucose Snf kinase inactive After determining that there was a osporylates Mig1 Mig1 goes to nudeus. URS element controlling glucose binds in a complex to repression at the Gall gene promoter, it was possible to go on to find the Mig1 protein that binds the URSGAL sequence(which turns out to lie in the promoter regions of many genes besides Gal genes). The Snf1 complex is a kinase that under low glucose conditions actively phosphorylates the Mig1 repressor, preventing it from entering theThe expression of β−galactosidase from each of these promoter deletion constructs under minus-galactose, plus-galactose, and plus galactose & glucose, are show. From these data we can deduce the location of cis-acting regulatory sequences for the Gal1 gene. • Deletions 7 and 8 do not express the reporter under any conditions because the deletions have removed some of the TATA sequence that is required for assembly of the basal transcription machinery. • Deletions 1 and 2 eliminate the ability of galactose to increase expression from the Gal1 promoter, and since expression is not induced there is nothing for glucose to repress. It turns out that the 75bp sequence between -310 and -385 is the DNA binding site for Gal4 and this kind of region is generally called a UAS (upstream activation sequence) and in this case UASGAL. We will come back to thinking about Gal4 binding to the UAS recognition sequence later. • Deletions 3, 5 and 6 have no effect on the ability of galactose to induce expression because the UAS remains intact. Note that shortening the distance between the UAS and the TATA region is not detrimental to induction. Indeed increasing the distance by inserting extra DNA between the UAS and the TATA sequence also has little effect on inducibility. This has led to the idea that UAS sequences can work at long distances (1,000 – 10,000 bp) away from the TATA sequence and the transcription start sites. (In mammalian cells regions containing binding sites for transcriptional activators are called enhancers; we will come to these in a later lecture) • Deletion 4 turns out to reveal information about glucose repression. For this construct, while galactose induces expression, glucose is unable to repress that expression. The deleted region defines the position of a sequence element needed for glucose repression, and a sequence element that behaves this way (i.e., are required for repression) is generally called a URS (upstream repressor sequence), and in this case URSGAL. No/low Glucose High Glucose nucleus nucleus cytoplasm cytoplasm P Mig1 Snf1 Snf1 kinase active, phosporylates Mig1, prevents nuclear localization Snf1 Mig1 Mig1 Snf1 kinase inactive, Mig1 goes to nucleus, binds in a complex to the URS No/low Glucose High Glucose nucleus nucleus cytoplasm cytoplasm P Mig1 Snf1 Snf1 kinase active, phosporylates Mig1, prevents nuclear localization Snf1 Mig1 Mig1 Snf1 kinase inactive, Mig1 goes to nucleus, binds in a complex to the URS No/low Glucose High Glucose nucleus nucleus cytoplasm cytoplasm P Mig1 Snf1 Snf1 kinase active, phosporylates Mig1, prevents nuclear localization Snf1 Mig1 Mig1 Snf1 kinase inactive, Mig1 goes to nucleus, binds in a complex to the URS After determining that there was a URS element controlling glucose repression at the Gal1 gene promoter, it was possible to go on to find the Mig1 protein that binds the URSGAL sequence (which turns out to lie in the promoter regions of many genes besides Gal genes). The Snf1 complex is a kinase that under low glucose conditions actively phosphorylates the Mig1 repressor, preventing it from entering the