Instead of looking for mutants that fail to execute glucose repression at the Gal1 gene, studies of the Gall promoter region itself provided the key to dissecting the mechanism of glucose repression Specifically the Gal1 promoter region was fused to the e. coli Lacz gene, on a plasmid that can GAL1 Lacz GAL1 AR galactokinase CEN galactose +galactose glucose B-galactosidase -galactose galactose + galactose glucose replicate autonomously in S. cerevisiae. It was first important to establish that regulation of Lacz (B-galactosidase) from the plasmid mirrored the regulation of Gal1 ( galactokinase) from its chromosomal locus;i. e. that B-galactosidase was induced by galactose in the absence of glucose but not in its presence. Having established that it was possible to go on and interrogate subdomains of the Gall promoter region for their role in induction of Gal1 by galactose, as well as repression of Gal1 by glucose. The minimal length of DNA stretching upstream into the promoter region from the gall transcription start site (designated as adjacent to-1 was 400bp DNA. Once this functional promoter region was delineated, systematic deletions 400 base pairs upstream of the Gall transcription start site is enough to confer of 50bp or so could be made all across the proper Gall-like regulation upon Lacz 400 bp region; this is easy to do with some recombinant dna tricks that are not important to know about here. suffice to 400-300-200-100 gl golgol &glu say that this"deletion analysis"revealed two regions critical for transcriptional control as well as the location of the tata al1 Promoter Gall Transcription sequence that is required for loading of the start site basal transcription machinery. Mapping GALl promoter elements B-galactosidase -400 300 200 00 -gal +gal +gal glu LacZ Lacz Lacz 2345678 LacZ LacZ Lacz Lacz UAS URS TATAInstead of looking for mutants that fail to execute glucose repression at the Gal1 gene, studies of the Gal1 promoter region itself provided the key to dissecting the mechanism of glucose repression. Specifically, the Gal1 promoter region was fused to the E. coli LacZ gene, on a plasmid that can replicate autonomously in S. cerevisiae. It was first important to establish that regulation of LacZ (β-galactosidase) from the plasmid mirrored the regulation of Gal1 (galactokinase) from its chromosomal locus; i.e., that β−galactosidase was induced by galactose in the absence of glucose, but not in its presence. Having established that, it was possible to go on and interrogate subdomains of the Gal1 promoter region for their role in induction of Gal1 by galactose, as well as repression of Gal1 by glucose. The minimal length of DNA stretching upstream into the promoter region from the Gal1 transcription start site (designated as adjacent to -1) was 400bp DNA. Once this functional promoter region was delineated, systematic deletions of 50bp or so could be made all across the 400 bp region; this is easy to do with some recombinant DNA tricks that are not important to know about here. Suffice to say that this “deletion analysis” revealed two regions critical for transcriptional control, as well as the location of the TATA sequence that is required for loading of the basal transcription machinery. 400 base pairs upstream of the Gal1 transcription start site is enough to confer proper Gal1-like regulation upon LacZ Gal1 Transcription start site Gal1 Promoter region 400 base pairs upstream of the Gal1 transcription start site is enough to confer proper Gal1-like regulation upon LacZ Gal1 Transcription start site Gal1 Promoter region 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8