nucleus. This situation (low glucose) is permissive for galactose induction of Gall gene expression via the UAs. In high glucose the Snf1 kinase is inactivated so Mig1 is not phosphorylated and the unphoshorylated migl enters the nucleus, to bind its URS sequence where it recruits two other proteins that together achieve repression of Gall expression Modular properties of Transcription Activators The Gal4 transcriptional activator turns out to be one of the most well studied proteins to carry out this kind of function. Once again a Lacz reporter was used in an imaginative way to establish that the Gal4 protein has two functional domains that are separated by a flexible region in the protein this but deletions are made across the Gal4 protein; the inverse of keeping Galg time the Gal1 promoter region remains intact upstream of the lacz report ntact and making deletions along the promoter, as described above Gal4 protein deletion analysis Essentially, if the N-terminal domain Lacz Reporter construct: of the gal4 protein is deleted, the protein can not bind to the UASGAL DNA sequence and so is unable to ctivation activate transcription of the reporter gene. But, in addition to DNA 门c binding Gal4 must have a region near the c-terminal end that is H responsible for recruiting and activating the RNa polymerase, thus allowing expression of the rep 口+艹」9ene, The most remarkable thing of all, was that a large region in the center of Gal4 can be deleted; as long as the dna binding domain is present at the N-terminus and the activating domain is present at the c-terminus Gal4 can activate transcription from the UASGAL sequence Gal4 missense mutations tend to map to the Activation of GaL transcription DB or the aD regions No galactose DNA binding Activation eD)Golly RNA Gal4- Recessive constitutive DB A D This remarkable separation of function between these two domains of Gal4 was dramatically demonstrated by a series of experiments called domain swapping. Essentially using recombinant dNa techniques, the Gal4nucleus. This situation (low glucose) is permissive for galactose induction of Gal1 gene expression via the UAS. In high glucose the Snf1 kinase is inactivated, so Mig1 is not phosphorylated, and the unphoshorylated Mig1 enters the nucleus, to bind its URS sequence where it recruits two other proteins that together achieve repression of Gal1 expression. Modular properties of Transcription Activators The Gal4 transcriptional activator turns out to be one of the most well studied proteins to carry out this kind of function. Once again, a LacZ reporter was used in an imaginative way to establish that the Gal4 protein has two functional domains that are separated by a flexible region in the protein. This time, the Gal1 promoter region remains intact upstream of the LacZ reporter, but deletions are made across the Gal4 protein; the inverse of keeping Gal4 intact and making deletions along the promoter, as described above. Essentially, if the N-terminal domain of the Gal4 protein is deleted, the protein can not bind to the UASGAL sequence, and so is unable to activate transcription of the reporter gene. But, in addition to DNA binding, Gal4 must have a region near the C-terminal end that is responsible for recruiting and activating the RNA polymerase, thus allowing expression of the reporter gene. The most remarkable thing of all, was that a large region in the center of Gal4 can be deleted; as long as the DNA binding domain is present at the N-terminus, and the activating domain is present at the C-terminus, Gal4 can activate transcription from the UASGAL sequence. Gal4 protein deletion analysis LacZ Reporter construct: Gal4 deletions: N- -C -C N- N- N- DNA binding lacZ UASGAL TATA lacZ UASGAL TATA N- DNA binding domain Activation domain LacZ activity + +++ - - + +++ + + + - -C + +++ Gal4 protein deletion analysis LacZ Reporter construct: Gal4 deletions: N- -C -C N- N- N- DNA binding lacZ UASGAL TATA lacZ UASGAL TATA N- DNA binding domain Activation domain LacZ activity + +++ - - + +++ + + + - -C + +++ N- -C DNA binding domain Activation domain N- -C DNA binding domain Activation domain DB AD Gal4 missense mutations tend to map to the DB or the AD regions Gal4- Recessive, uninducible Gal481 Dominant, constitutive N- -C DNA binding domain Activation domain N- -C DNA binding domain Activation domain DB AD Gal4 missense mutations tend to map to the DB or the AD regions Gal4- Recessive, uninducible Gal481 Dominant, constitutive G a l8 0 D B A D G a l8 0 D B A D This remarkable separation of function between these two domains of Gal4 was dramatically demonstrated by a series of experiments called domain swapping. Essentially, using recombinant DNA techniques, the Gal4