Genetics Lecture No. 18: Eukaryote Gene Regulation II
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.
-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
◦ 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