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17 Cards in this Set
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Responsible for Gal (Galactose) metabolism. So we're not going to want to turn on the promoter unless Gal is present so we can import more galactose and utilize this by breaking it down |
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We have several players here. Gal4 is always bound to the UAS region. Gal4 is a transcription factor that functions as a dimer. We need 2 copies of Gal4 in order for it to function. |
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Gal80 |
We have Gal80 which is a repressor. It is also normally present. We have gal4 ready to activate, it's function is repressed because gal80 blocks its activation domain. So if we didn't have gal80 here, that would be fine, and we could induce expression of the gal1 promoter. But because gal80 is always expressed, just like gal4, this is the normal situation when we don't have any galactose present. |
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Gal3 |
We have a 3rd factor called Gal3. Gal3 is sequestered in the cyto until gal is present. So it won't be able to enter the nucleus and influence gene expression unless it's bound by a molecule. Unbound gal3 can't enter the nucleus, just hanging in the cyto nonfunctional. When gal enters the cell, it can bind gal3 to change it's shape to let it enter the nucleus. When we have all 3 of these factors together (gal1, gal3, gal80), gal3 makes it so that we have re-exposed the activation domains of gal4. |
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Another way to help enhance transcription at promoter region... |
There are multiple gal1 promoters throughout the cell. Some might be around nucleosome (like in heterochromatin). We might need to loosen up the DNA around these regions for this. In this case, we might need to recruit a histone-acetyl transferase, in this case the HAT is specifically called SAGA. We can get saga recruited when we have all 3 factors present. It puts acetyl groups on lysines of histone tails that are near where the UAS is. We acetyl lysines on histones here which will recruit bromo-domain containing proteins that can bind and help remove gal1 sequence from being wrapped around a nucleosome. |
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B. Gal1 would never be expressed. Gal4 has the activation domain Gal80 blocks the activation domain Gal3 removes Gal80 so the activation domain is present |
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We make synthetic proteins by taking an activation domain of 1 protein and fusing it to the DNA binding domain of another protein. We can take the DNA binding domain and the activation domain of gal4, split them and fuse them to other proteins. In this case, the purpose is to identify another transcription factor that induces gene expression or looking for protein-protein interactions. If proteins X and Y interact with each other, they will bring together the binding domain and activation domains of Gal4 so that we can get expression of a gene. We'll use a reporter gene in this case. A reporter gene is something that can be like a fluorescent protein that will be coded for, because it's easy to detect so we don't have to do a western blot if we did a regular gene. So if we see the reporter gene being turned on that means we have successfully brought together both gal4 domains. This only happens if the 2 proteins we are studying interact with each other. |
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Take 1 yeast strain that has 1 fusion protein and mix it with another yeast with the other fusion protein. They will mix and if we see a fluorescent yeast, that means the proteins interact. |
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Don't need to know factor names. We have different yeast mating types (haploid cells, diploid cells etc). The two haploid cells can fuse to make a diploid a/alpha cell. We're going to want to express a different set of genes if we're the a cell, the alpha cell or the diploid cell. The transcription factors of these 3 types come together to regulate gene expression. |
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This is what we call "the mating type locus." This is going to encode transcription factors that are specific for either cell type. 1 mating type locus will make the transcription factors alpha 1 and alpha 2. The other one is going to make a1 only. Depending on whether alpha1 and alpha2 or a1 are being expressed, they can each bind a transcription factor called MCM1, which is expressed in common. Depending on whether mcm1 is bound to a1 or alpha1 or 2, we get different types of expression. |
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A) Lets say we have an a type cell. This is going to make a1 and mcm1. For the a cell, mcm1 can bind the promoter region for the a specific genes and induce gene expression. The alpha specific genes require a factor to turn them on that the a cell doesn't have, so the alpha promoter region is going to be naturally off. The haploid specific region is naturally on in these cells. We actually only need mcm1 to turn on the a specific genes and the haploid genes, and turn off the alpha specific genes. B) In the alpha cell we make alpha1,2 and mcm1. The combo of these 3 things makes a heterotrimer. This is a transcription factor that only functions with these 3 things. This turns off the a specific genes. C) When the a and alpha cells mates, they can still make a1, alpha 2 and mcm1, but they can no longer make alpha1. |
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Reminder that we have 2 domains |
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We can have different combos of these domains to regulate transcription. They will typically bind an inverted repeat sequence somewhere on the DNA to enhance gene expression. Each of the 3 dimers will recognize a specific sequence. So with just 2 different proteins, we can recognize 3 different sequences on a DNA. With 3 proteins, we can recognize 6. |
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If something is bound to the insulator and it's in between the enhancer and promoter, it will block it's effects |
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Example of how hormones are regulating gene expression |
In the gal1 promoter, we had a factor that was in the cyto until gal was present. This is also true for many hormone receptors. We can have a signal outside of the cell, like a nutrient or growth factor, that binds a receptor, the receptors will phosphorylate each other, which phosphorylate other factors which can only enter the nucleus when they are phosphorylated. Phosphorylated mapk can then enter the nucleus. At any one of these steps, we can have regulation. So these steps will want to be highly regulated. |
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Steroid hormones involve receptors that are in the cytoplasm until their cognate hormone is available. When that hormone binds the receptor, the receptor changes it's shape in a way that it can enter the nucleus. So here we have a steroid hormone that can diffuse into the cell. The receptor for that hormone is bound to another protein where it can no longer enter the nucleus. Once that steroid binds the receptor, it has the ability to enter the nucleus, bind dna and trigger gene expression responses. The elements that steroid hormone receptors bind are called hormone response elements. |
