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Lecture 11

BIO240 Lecture 11

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University of Toronto St. George
Jennifer Harris

Lecture 11: Protein-protein interactions - Let’s take stock: we’ve talked about the transcriptome, its involvement b/w organisms even within an organism. We’ve talked about transcription factor use, which gene regulatory proteins are used at which points in time & we’re going to determine ultimately what the transcriptome is that gets made in those cells. We took a look at the fact that the proteins that ultimately get made from the transcriptome is going to give rise to those differences – those proteins don’t function in isolation (E2-E3 ubiquitin ligase complex – 2 proteins working together to perform an important cellular function). Proteins are functioning together in complexes that really drives differences b/w cells which is what we’re going to focus on in today’s lecture. Interactome – interactions b/w proteins Complete set of protein-protein interactions produced by the proteome at any one time. Yeast 2-hybrid assay - Helps us define, not just a few interactions b/w proteins but a good many interactions b/w proteins. In fact, all that occur within cells. - Stan Fields developed the yeast 2-hybrid assay. - The yeast two-hybrid assay takes advantage of the way in which eucaryotic transcription factors/gene regulatory proteins work. Identify binding partners – 2 proteins that interact with each other. - Looking for the interaction b/w the BAIT protein & its corresponding PREY, can be thought of protein 1 and protein 2 & testing the hypothesis do those proteins interact with each other? - System takes advantage of a transcription factor that has been split in half, DNA binding domain on BAIT & transcriptional activation domain on PREY separating those 2 activities so the protein no longer functions as one protein independently but requires those 2 domains to be brought together by the interaction b/w the 2 proteins to eventually activate transcription. How does that all work? - Starts with transcription factor called GAL4. - GAL4 has a DNA-binding domain DBD and a transcription-activation domain. - UAS G upstream activator sequence. - So when galactose is present, GAL4 binds to this upstream activator sequence that is found upstream of a minimal promoter & it activates a gene encoding galactose utilizing enzyme so there are coactivators and RNA polymerase II, have the mRNA made and the corresponding enzyme. - Like other eucaryotic transcription factors, it turns out that the DBD & the TAD are separable & can function on their own, that is, the DNA binding domain can bind DNA in the absence of the activation domain. The activation domain provided that it is tethered to a DNA binding domain will function as an activation domain. It turns out that the activation domain needs to be somehow be associated with the DNA binding domain to do its job. Bait Prey - They identified by doing a number of deletions what portion of the sequence was the DNA binding domain so they know that DBD must reside upstream of this particular sequence b/c if deleted, it loses DNA binding activity. By deleting from the carboxy terminus, they identified similarly the region that’s necessary for transcription (TAD). - They’ve done internal deletions, & shown that by reducing proteins to this little stretch of 74 AAs here & a little under 120 AAs, that you have both DNA binding activity & transcriptional activation showing that those 2 bits are the important bits. - For the yeast 2-hybrid assay, what’s happened is that these 2 pieces have been separated so that you still have DNA binding that can occur b/c you have the DNA binding domain but b/c the transcriptional activation domain has been separated from it, there is no longer transcriptional activation. What’s been done is to fuse a BAIT protein, one protein of interest to the DNA-binding domain & a so called PREY protein in translational fusion with the transcriptional activation domain – if the 2 proteins interact, they will bring the DBD in close enough proximity to the TAD so now we acquire DNA binding activity & transcriptional activity & we’re able to activate a corresponding target gene. - If BAIT and PREY interact they bring the DBD & the TAD into close enough proximity that they’re able to activate transcriptional activity from genes containing the UAS . G - All thesestomponents are encoded on plasmids. - In the 1 instance, we’ve got a promoter driving the expression of DNA that encodes the GAL4 DBD & this is upstream of our gene of interest that encodes our protein of interest, the BAIT. Together they’re going to make a fusion protein which is the BAIT comprising the DBD plus the protein of interest. - In yeast, in order to make sure that our plasmid is there, we need to include an extra gene so it’s a promoter driving the expression of the TRP gene. Selectable marker - Yeast cells with the plasmid b/c they have the TRP gene on the plasmid will enable the cell to make tryptophan & you don’t need to have tryptophan in the medium. - This way we make sure that only cells that have the plasmid are the living cells – the living cells are the only cells that can possibly have the plasmid – make sure that our plasmid is there making our fusion protein. - TRP gene is a selectable marker that ensures that we’ve got a plasmid present that is going to make our BAIT. - Fusion protein comprising our protein of interest (protein 1), our prey and the GAL4 activation domain. Need another gene to select for this particular plasmid that has a promoter & the functional LEU gene. Complemented – the absence of Leu & Trp is complemented (filled in) by the presence of the Trp gene on the BAIT plasmid & the Leu gene on the PREY plasmid. - Now when one sets this up, they’re interested in asking what are all the different proteins that our bait protein interacts with? - In one yeast strain, set up the bait plasmid plus one prey plasmid & in the next yeast strain that you have, you have the same bait plasmid but a different prey plasmid, a different protein, & so on b/c what you’re asking is sequentially what are the all the different possible proteins that my first protein can interact with. - Create a library of different proteins then that are all different possible preys – which of those interact with our bait to bring these 2 components together. - Yeast cells are transfer with bait & prey plasmids so that goes into the yeast cell to complement the mutation that is present there, the double mutation of Trp & Leu. Reporter gene - Reporter gene has 3 components to it: upstream activating sequences to which the GAL4 binding domain binds, a minimal promoter to support transcription by RNA polymerase II & the actual reporter itself, the His gene. - If the cell has the plasmid & if there’s interaction b/w the 2 proteins, the HIS gene will be activated & the cell can survive in medium lacking His, Leu & Trp. 1) When the proteins interact 2) Transcription of His occurs - When interaction occurs, have activation of the His gene so cells will survive in medium lacking Trp, Leu & His. - If there is no interaction b/w the bait and prey, then the cells will die in that particular medium. - The interaction b/w those 2 proteins bring the DBD & the TAD into close enough proximity that transcription is activated. **Lecture 11 Ends Here** **Lecture 12 Starts** - Today we will have a recap that carries us all the way from genome to phenome. - Last lecture we were taking a look at the proteome and how different proteins within the proteome interacted with each other and it's this interaction between proteins to give rise to the interactome that define differences between cells tissues and organisms. - What we looked at in the last lecture was a mechanism by which we could use, the power of yeast genetics and our knowledge of transcriptional regulation to test for the interaction between two proteins. If we hypothesized that 2 proteins interacted with each other, we could make use of the yeast-2 hybrid assay. This yeast 2 hybrid assay is used to assess protein-protein interactions whether a particular bait protein can interact with the corresponding prey proteins, whether these two proteins have the capacity to interact and this is defined by the yeast-2 hybrid assay. - Recall that we dissected the DNA binding domain function of the GAL4 transcriptional activator from its transcription activation domain and through genetic engineering we created a fusion protein between the GAL4 DNA binding domain and our bait protein and the GAL4 activation domain and the prey protein and the interaction of the two proteins then the bait and the prey would bring the DNA binding domain close enough in proximity to the dissected transcriptional activation domain to activate transcription at a target reporter gene bearing the upstream activating sequences that GAL4 binds to so the UAS for GAL4, a minimal promoter and then a reporter gene, the Histidine gene. - We looked at/said cells live wherever there is this interaction because the yeast cells live, wherever there is this interaction because the yeast strains are grown in a medium lacking leucine, trp and his and each of the two plasmids is able to complement the absence of the trp and the Leu function, the synthesis of tryptophan and leucine so in order for cells to live they must have both of those plasmids and since the medium is lacking histidine, it must be able to transcribe the His gene to give rise to the ability to make the AA histidine. 1)  Positive 2)  Negative 3)  Negative 4) (both false positive and false negative) 5)  Negative 6)  Positive - The yeast two hybrid assay is prone to some artifacts. Recall that what we’re doing is very artificial, we’re asking can protein X interact with protein Y to bring the GAL4 activation domain together with the GAL4 DNA binding domain. So there are some artifacts that can arise when we use this system to assess protein-protein interactions. - First of all, imagine that the bait protein has a transcriptional activation function, it's
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