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Lecture 18

Lecture 18: "Eukaryotic Gene Regulation II"

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Biology 2581B
Jim Karagiannis

Genetics Lecture No. 18: Eukaryote Gene Regulation II th Monday March 18 , 2013 Silencers & Repressors: -In eukaryotes, whenever the repressor binds a silencer, transcription of the gene is stopped. Repression can override activation (there is no transcription of the gene, even if the activator is bound to the enhancer). Repressors are proteins that bind specific DNA elements and inhibit transcription. They block or destabilize RNA polymerase assembly and movement. Repressors can also act by interfering with the function of activators through competition and quenching. Some repressor proteins act by competing for the same enhancer elements as activator proteins. But repressor proteins have no activation domain, so when they bind to enhancers, no activation of transcription can occur. A second class of repressors act by binding directly to the activator proteins themselves to quench activation in one of two ways. Type I quenching is achieved when the repressor prevents the activator from reaching the enhancer. Type II quenching is achieved when the activator can bind to the enhancer, but the repressor prevents the activation domain from binding to basal proteins. Model Of Yeast Transcriptional Regulation In Galactose Metabolism: -The ability to engage in genetic analysis is a crucial advantage to using the budding yeast Saccharomyces cerevisiae. This unicellular eukaryotes has two mating types: the a haploid and the α haploid. When existing in the haploid state (1 copy of each chromosome), the yeast can be induced to mate (fusing through the process of conjugation) with another haploid by limiting nutrients. After inducing a diploid yeast cell to sporulate, we can make double mutants by taking an α haploid with X mutation and allowing it to mate with an “a” haploid with Y mutation. The resultant diploid is induced to sporulate, whereby the spores will show wild type, X mutant, Y mutant and double mutant phenotypes. -The inducible (by the presence of galactose) expression of the GAL7, GAL10 and GAL1 genes is required for yeast cells to use galactose as a carbon source. The deletion of cis-acting elements in the upstream regulatory regions (defined as an enhancer element that acts on all three genes) gives basal metabolic rates. Looking for mutations that affected the regulation of the GAL genes, it was discovered that a loss of function mutation in another gene, GAL4 (GAL4 ) prevented galactose from activating expression of the three genes. Testing Alternatives: -GAL4 (activator) binds to the enhancer increasing the transcription of GAL7, GAL10, and GAL1. -GAL80 (repressor) binds resulting in constitutive expression of GAL7, GAL10, and GAL1. -How do GAL4 and GAL80 interact with one another in the pathway controlling transcription of GAL genes? ◦ Alternative #1 (Galactose inhibits the inhibitor GAL80): Galactose ----| GAL80----| GAL4 ---→ GAL7, GAL10, GAL1 Transcription ◦ Alternative #2 (Galactose inhibits the inhibitor GAL4): Galactose ----→ GAL4 ----| GAL80 ----| GAL7, GAL10, GAL1 Transcription If you lose GAL80, there is no check on transcription (really high levels); losing GAL4 (inhibition of inhibitor) results in low basal levels. In both cases, galactose positively inhibits the inhibitor resulting in high transcriptional levels. -Creation of GAL4 / GAL80 double mutant can distinguish between the two alternatives: - - If Alternative #1 is true, then we predict the GAL4 / GAL80 double mutant to display basal levels of transcription because the GAL4 phenotype wins out. - - If Alternative #2 is true, then we predict the GAL4 / GAL80 double mutant to display constitutive expression because the GAL80 phenotype wins out. - - -The results of conjugating a haploid GAL80 mutant with a haploid GAL4 mutant revealed basal levels of transcription, demonstrating the validity of Alternative #1 (GAL4 is more downstream in the pathway). Biochemical Evidence For The Yeast Model: -The GAL4 gene-product has two domains: a DNA-binding domain which recognizes the enhance
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