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Lecture 18

BIO240H Lecture 18.doc

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Department
Biology
Course Code
BIO120H1
Professor
Jennifer Harris

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Lecture 6: Eukaryotic Transcriptional Regulation I Slide 1 - Today we talk about proteins involved in chromatin. We have a shopping list of crap to go through, revisit prokaryotic and eucaryotic transcription, talk about activator proteins. We aren’t actually going to get the latter part and that will be in the next lecture. Slide 2 Slide 3 Is packaged into chromatin Is unable to initiate transcription on its own Act at a distance residing - Now he comes back and makes this statement telling the truth, peeling away the onion and seeing the complexity that resides. There are 5 different points that underlie differences and complexity between bacteria and eucaryotic gene regulation. There are actually 6 different points but in the textbook they only mention 5, the sixth one is the nucleus so that’s another possibility for regulation and that’s an important point. - Point number 3 is probably something that is evident to us, there is a lot of DNA in eucaryotes. We know there are a # of contexts under which many genes must function – where a gene must be on in your lung at some time during development and then in your brain at another point in development. One of his favorite examples is housekeeping genes in animals function throughout the body giving rise to enzymes that have housekeeping functions like enzymes involved in glycolysis. Some of those same proteins are transcribed at a huge amount resulting in a huge amount being made and they make up the lens of your eye. There must be differential levels of regulation and must occur in different cell types so it implies that the switches that reside next to a gene that say transcribe here or transcribe this amount, must be pretty large at least to bacterial sequences. This is point 3, eucaryotic gene regulatory sequences act at a distance, thousands of nucleotides away from the minimal promoter sometimes therefore many possible regulatory sequences can reside in between. Slide 4 - The first is that we have nuclear import as a possibility from gene regulation, not talked about in the textbook b/c it’s separated nuclear import from gene regulation. The first one is that once you get into the nucleus, you have chromatin so eucaryotic DNA is packaged into chromatin and as we seeing the remainder of the lecture that provides regulation opportunities that aren’t available to bacteria. Because of the way gene regulatory proteins function, the sequence specific DNA binding proteins bind at specific regions, read the DNA sequence, bind to these motifs and they affect the histone code themselves. It is these sequence specific DNA binding proteins and their interaction that is going to remodel chromatin, an opportunity that just doesn’t exist for bacterial gene regulation. Slide 5 - Point number 2 is that eucaryotic RNA polymerase II which makes RNA is unable to initiate transcription on its own, it's a wuss and can’t do its own job, needs helpers and we know that from Professor Chang’s lectures. We know RNA polymerase II needs a set of general transcription factors and therefore we have multiple steps available depending on which transcription factors are there, the rate at which they are recruited, the rate at which they get retained in the complex will determine ultimately the ability to initiate and sustain transcription. - This is going back to Professor Chang’s lectures: remember there are many rate limiting steps not the least of which is that enzymatic reaction are catalyzed by components of basal transcription machinery and as an example; the phosphorylation of polymerase II by transcription factor 2H and this coupled with the availability of those transcription factors and the ability to recruit them is going to provide as the slide says. - A number of rate limiting steps that can function as transcription control mechanisms. He wants to underline the fact that it can control the timing and localization of initiation, the extent to which expression occurs (lot of transcription or a little) and the duration to which it is sustained. - The importance of assembling everything together is point number 2. Slide 6 Control gene expression from a distance - Let’s take a look at the implications related to point 3. - This serves an important function not only to provide a number of switches near a coding sequence, doesn’t have to be upstream, it also ensures that protein-protein interactions can occur by having things at a distance b/c DNA isn’t perfectly bendy, it is flexible but not great. If you tether two proteins to a stretch of DNA shown by the white circle and the red circle, and the DNA is shown in red is that red line. If we tether those two proteins having them between 500 to 5kbases apart, they can interact with a high degree of frequency because DNA is that bendy. - This is shown nicely in the upper and bottom panels. The top panel shows 2 proteins tethered to DNA and the blue cloud that is around it indicates the frequency of interaction – can see that there is a high degree of interaction right adjacent to each other in this cloud. If we tether the two proteins only 100 base pairs apart, there seems to be a zone of exclusion there because DNA can’t bend back that tightly on itself. - This can be captured in a graph showing things drop off precipitously certainly under a 100 ties apart, then there’s an optimal length around 750 nucleotides apart where proteins can interact with each other with a high degree of frequency. - So binding of proteins to different sites on DNA will cause interaction frequencies to increase as you reach this optimal distance. So the intensity of the blue cloud indicates the likelihood of collision and the frequency of collision is increased down to 100 bp after which collisions are restricted. - Also multiples of ten base pairs are important and why? They are important because they place them on the same side on the double helix, putting them in the major groove. We tend to find that spaces between binding sites are in multiples of ten. - We have the capacity for proteins to bind at a distance that function to regulate the function of RNA polymerase II at the minimal promoter. Slide 7 Control gene expression from a distance - Captured beautifully in this slide from the old textbook and we can see the binding at a gene regulatory site by an activator protein and we see the gene expression is controlled from a distance that can be hundreds to thousands of base pairs away from that minimal promoter where the TATA box is. - The role that it's playing in this regard is to recruit additional proteins to the minimal promoter to actually activate gene expression. - What are the sequences that are there? Slide 8 Promoter and regulatory DNA sequence Upstream and downstream of coding sequences Gene binding proteins - These gene regulatory sequences comprise: the minimal promoter + additional gene regulator sequences. - We’ve learned about the minimal promoter already (TATA box & surrounding plot) that allow RNA polymerase to bind and do its job. - But we need in addition to that are sites that recruit gene regulatory proteins. Most of them are upstream of the minimal promoter but holy crap look there’s one downstream too, not only downstream of the TATA box but actually downstream of the whole coding sequence. This tells us that gene regulatory sequences can reside both upstream and downstream of the coding sequence. - How could that be true? How can you have gene regulation occurring backwards? It's always 5’ to 3’. How can you have gene regulatory sites downstream and functioning in a promoting capacity? Remember DNA is arranged in a 3D structure and chromatin, something we think of as linear for downstream, may in fact in terms of chromatin be right next door. The gene regulatory protein may actually be right beside it and what we want to do is to change the local structure of chromatin around there so RNA polymerase can get on and do its job. - Not only is it downstream or upstream, it can be in introns as well. Introns (junk DNA) can have regulatory gene sequences there, it can function in important/crucial gene regulatory role as we will see in the next lecture. - All of these are target binding sites for gene binding proteins. Movie - We’re looking inside a cell – zoom into nucleus, look at DNA and a minimal promoter to start with. TATA binding proteins bind to TATA box. DNA is bending, & very slowly it is going to recruit basal transcription factors and eventually RNA polymerase II. Eventually the DNA will unwind slowly. Eventually RNA polymerase will do its job. - This is a very simplified version of what’s truly happens b/c what need next door are activator proteins binding elsewhere in the DNA and they will eventually recruit the TATA binding proteins. The subsidiary transcription factors come and here we go again. - This takes us to the end of point 3. Slide 9 - Recall that eucaryotic genes are not organized into operons – they are individual genes, gene regulatory region, coding sequence and that’s how they function. They can be co-regulated, that is they are transcribed at the same time but they are never located right next to each other to create a polysystronic message – no operons so independent regulation. - Let’s take a look at those gene regulatory proteins that do the job. Slide 10 Modular - He wants to focus on eucaryotic gene activator proteins. The same is a little true for repressor proteins but some themes are different. - Tend to be modular in structure – this means they have different modules, all one protein, 2 different bits to the module like having an arm up there and a leg down there. They are all part of the whole but perform different functions. - For transcriptional activator proteins, they have a DNA binding domain that binds to a recognition site and a transcriptional activation domain. These two domains can be completely separated from each other and their functions shown separately. - Let’s show an experiment in the bottom two panels. What we’ve got in this example is the Gal4 protein with its DNA binding domain which can bind to a recognition site, the activation domain is able to activate transcription at the minimal promoter and turn on a particular reporter gene, lac Z in this particular instance. - If we take the transcriptional activation domain and create a fusion protein with a different DNA binding domain, it no longer is able to bind to its binding site and obviously doesn’t turn the corresponding downstream gene on. - If by contrast we fuse it again to a different DNA binding domain but then make that DNA binding domain available on a stretch of DNA upstream of a minimal promoter, the transcriptional activation activity is able to be recruited to a new site, the one bound by lac A in this instance, activate gene transcription and turn on the lac Z gene. Slide 11 - So what is it doing when it's doing that? That activation domain is increasing relatively weak and nonspecific interactions between the DNA and RNA polymerase holoenzyme. It's grabbing the RNA polymerase holoenzyme and saying you need to be here. It increases it up to a thousand fold, that interaction. It can do this by recruiting specific members of the basal transcription machinery and a really important protein complex that is your blank on the next slide. - It can do this by recruiting the mediator protein complex. The mediator protein complex is a multi-protein complex, 20 different proteins that function to assemble the pre-initiation complex of RNA polymerase II at the minimal promoter. - That’s shown nicely in two slides. Slide 12 The mediator - This is the figure from last year: here is the transcriptional activation protein bound to its activation site with the activation domain and it will recruit through protein-protein interactions, the mediator which then assembles the RNA polymerase II transcriptional apparatus at the minimal promoter. - This year, it shows 4 different protein complexes coordinating the mediator at the same site. It shows how all these different gene regulatory sequences can reinforce the interaction of RNA polymerase II with the minimal promoter. Slide 13 - What else is going on at that point in time? Recruitment of our old friends, the chromatin remodeling complex which can increase accessibility to the TATA box just by remodeling the nucleosomes. It can remove the nucleosomes altogether resulting in great interaction with the TATA box. It can change the histones that are present in the nucleosomes again increasing the interactions with TATA box and finally it can recruit histone modifying enzymes that can modify the histone code. - That is our focus for the final point. Slide 14 Lysine Neutrally Repel/Disassociate - This takes us back to our histone code. Recall there are multiple sites for post transcriptional modification along the amino terminal tail. Recall that histones can be acetylated on their amino terminal tails – on the lysine residues making them more neutral. And resulting (and he doesn’t like the textbook’s saying) in repelling of DNA from the histone proteins – it's not really repelling, it's just not associating as well so it's looser not really pushing apart per say, it's just not as tightly bound. The ultimate result is that you end up with an alteration of the histone code resulting in gene expression. - He wants to walk through an example in the last bit. Slide 15 - Imagine we have a nucleosome, we recruit a DNA binding protein to it, and that DNA binding protein can do a # of things itself – recruit rewriter proteins, recruit acetyl transferase, recruit histone kinase, adding acetyl groups and phosphate groups to the histone which are then recognized by chromatin remodeling reader proteins, TFIID & we end up with initiation of transcription. Slide 16 - This is a flow chart of what he’s been talking about. - What happens with tra
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