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Lecture 17

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Jennifer Harris

Lecture 5: Transcriptional Regulation 3 Slide 1 - Next lecture we’re going to be looking at eucaryotic transcription regulation. Today we compare prokaryotic and eucaryotic transcription regulation. - Last Lecture: Last time we looked at the components of genetic switches: small DNA motifs and moved onto protein motifs that interact with the small DNA motif that together comprise genetic switches. - This lecture: We’re going to see how these switches function in vivo. - Last lecture: You can document interaction between DNA and protein in vivo using chromatinimmunoprecipitation protocol. Today we see how these work in vivo. - We looked at the interaction between protein and DNA – the genetic switch in vivo. Let’s take a look and think about how they might have to work in vivo to fulfill their purpose of turning genes on and off. Slide 2 - Simple organisms to begin with: prokaryotes. They have simple development with one single cell type, they can become an elaborated cell form but overall simple cells. These cells must respond to fluctuations in the environment & perhaps no greater fluctuation exists than that in resources that are available for the organism to grow and survive. As a consequence one of the things that occurs is that these microorganisms/procaryotes are incredibly motile, they can move through media to search/savage out resources they need to grow divide and reproduce asexually making progeny daughter cells. - Movie: the cells are moving rapidly through medium to bind to those particular resources. The end result is a colony that has grown out and scavenged effectively for resources in the medium in which it is growing, foraging for resources. - What they must do is to respond to environmental cues and to the resources that are available to them and respond appropriately so either to move in the proper direction to get those resources or to modify their metabolism to acquire those resources and use them when they become available. They all depend on changes in gene regulation. Slide 3 - Bacteria have evolved a very simple system for responding to environmental cues. You have a nice simple transcriptional model, there is no nucleus so signals can move freely to the DNA invoking transcription and then translation – very simple regulation of gene expression. - This is simplified further by clustering groups of genes with similar functions or that function in a sequential manner, for example in a metabolite utilizing pathway or a metabolite biosynthetic pathway, organized so that the coding sequences are all under the control of one simple DNA switch and this is known as an operon. Slide 4 Continuous genes Single RNA molecule Single promoter - An operon is a stretch of DNA found on a bacterial chromosome where you have a group of contiguous genes (coding sequences all in a row) that are transcribed simultaneously into a single RNA molecule under the control of a single promoter. - Example of a trp repressor, the trp operon functioning together and we’re going to look at the trp operon. - Here we have it illustrated schematically so this is a segment of the E. Coli genome and you can imagine the chromosome continuing on to make up that large circular chromosome that makes up the E. Coli genome. - What we have in a row as promised is a contiguous stretch of genes, coding sequences for genes E, D, C, B and A. We can see that they are all under the control of one simple genetic switch that resides upstream of these coding sequences. The promoter itself comprises of many different motifs of operator sequences and other small gene regulatory motifs we won’t focus on today. We will focus on the operator site which mainly functions as an on/off switch for the particular operon, allowing the transcription of those coding sequences. Produced into 1 RNA that is polysystronic in that it has a number of systrons or coding sequences arranged adjacent to each other in one contiguous stretch of RNA. What happens is that the polysystronic RNA is translated into the 5 gene products – the gene products are used in the biosynthesis of the amino acid tryptophan hence the name trp operon. Slide 5 Ligand binding determines interaction with DNA - The way this operon functions is to be responsive to the availability of Trp in the growth medium or in the environment surrounding the E. Coli. - In conditions where concentration of Trp is low, there is a protein which normally functions to repress the activity of the operon that is unable to bind to the operator sequence. Think of the operator as the switch and the trp repressor is the gene regulatory protein that turns that operon off. Under low concentrations of Trp, it is unable to bind, unable to repress the activity of the operon. - In the presence of Trp, this binds to the trp repressor and as a consequence the trp repressor binds to the operator site so it's the binding of the ligand of Trp that determines the interaction between trp repressor and the operator site. The small molecule/ligand is what determines the interaction between the trp repressor and the operator site, its DNA target, so ligand binding determines the interaction with DNA - You shouldn’t think of it just as an on switch but also as an off switch. If the Trp concentration was to drop again, there wouldn’t be Trp around for the repressor to bind to so the repressor wouldn’t bind to the corresponding operator site. - That is how this simple switch works: ligand there, repressor binds, ligand isn’t there, repressor doesn’t bind. When Trp is present, the repressor binds and you don’t need to make Trp biosynthetic enzymes at that particular point in time and it's perfect but when Trp isn’t around, the repressor no longer binds and you get the transcription of downstream targets and you’re able to make Trp. Slide 6 Transcription OFF De-repression Transcription ON - High concentration of tryptophan, repressor is bound and as a consequence RNA polymerase (blue blob) is unable to initiate transcription so transcription is correspondingly off & you get no synthesis of tryptophan as a consequence. You don’t need to synthesize tryptophan when you have it around. - By contrast when tryptophan concentrations are low the repressor is unbound and the RNA polymerase is able to bind to the promoter initiating transcription so it's on, and a cell which is now not having tryptophan available from environment, will synthesize it on its own. - This process in which Trp is absent so you now have transcription being on is called a system of de-repression. So repressed in presence of the ligand and de- repressed in the absence of the ligand. Slide 7 Repression/de-repression Negative regulation Regualtory protein = can also Inducer (de-repressor) called repressor/activator Co-repressor Ligand = acts as substrate - We can have what is known in the first instance as negative regulation and that is when a bound repressor protein prevents transcription so it is negatively regulating the gene in question. - Here in the first example we have a bound repressor protein and in the presence of an inducer which can also be seen as a de-repressor it binds to the corresponding repressor and causes it to move away from the DNA. The ligand switches the gene on by removing a repressor – this is de-repression and that is precisely what we saw with the trp operon and the trp repressor is this negative regulation involving inducer/de-repressor. - By contrast, you can have what is known as a co-repressor binding together with repressor and the binding of this particular ligand allows the gene regulatory protein to bind to the DNA resulting in co-repression, they’re working together to repress the gene so ligand removal in this instance switches the gene on by removing the repressor. (I think he made a mistake because co-repression is when the ligand which is tryptophan joins with the repressor to repress the activity of the gene, it's not a de repression, please let me know if he announces anything about it) - In both of these instances what we have are examples of negative regulation, the binding of repressors that are influenced by the presence or absence of the corresponding ligand to which they bind. Slide 8 Activation / deactivation Positive Regulation Inactivator Inducer (co-activators) - By contrast, we can have as opposed to repression/de-repression, we can have activation/deactivation as a common theme. This is where the interaction between a ligand and a DNA binding protein resulting in the activation or deactivation. This is referred to as positive regulation because it involves the switching on of genes by the binding on of gene regulatory proteins - Here what we have is an activator protein that promotes transcription as opposed to a repressor which represses transcription. - Let’s take a look at these examples right here, we have inactivator ligand in the first one that normally the activator protein is able to bind to the promoter and promote transcription but in the presence of an inactivator protein, this positive gene regulatory protein no longer is able to bind so the ligand binds to remove the regulatory protein from the DNA and switches the gene off by removing the activator and this is deactivation. - Alternatively we can have the ligand functioning as an inducer. In this instance, the gene regulatory protein binds only in the presence of the inducer and ligand removal switches the gene off by removing the activator. Or to switch it on the other way to view it is it functions as a co-activator. - So how to distinguish between the two slides? Negative regulation involves repression proteins, those switch genes off and positive regulation involves activator proteins, things that will switch the gene on. The positive regulation versus negative regulation can be influenced one way or another by either co-repressors/co- activators, de-repressors/deactivators (inactivators). Slide 9 - How do these function in a real context? - We will move away from how ligands function to regulate prokaryotic gene expression to look at an example or two to effectively further flesh out these switches by looking at how some proteins can function as both activators and repressors. - In this particular instance, size does matter – there is a context dependent role and that is the distance between where the protein has bound and where the binding site for RNA polymerase is, is very important to whether the protein is going to function as an activator or repressor. - So with lambda repressor, if we have one promoter, if the distance is appropriate distance between operator and promoter binding site, transcription can occur so the distance between the operator & promoter allow the lambda repressor to promote RNA polymerase binding. - By contrast at another promoter, the lambda repressor actually functions as a repressor where the distance between the operator site and the promoter site is such that it inhibits the binding of RNA polymerase and therefore prevents downstream transcription of this particular gene. - Note that the distance in the first is a good one allowing the recruitment of RNA polymerase and the other instance, it actually inhibits the binding of RNA polymerase. - This is very simple to either recruit or inhibit the ability of RNA polymerase to do its job to transcribe the downstream sequences. - If you look at this figure in the textbook 7.38, you will find that he’s done something where he’s made sure that he is showing transcription as occurring off the right hand side, but in fact in the textbook they have this in the reverse direction and the reason he showed it this way is how these genes function in vivo, that is the lambda repressor binds at one site and one promoter goes in one direction and when it's bound it allows promotion of transcription in this direction and prevents it in the opposite direction. - There are conditions we will talk about that cause the lambda repressor not to bind but then allow the transcription of the gene that goes off to the left hand side. Slide 10 - We have seen a bit of this like in the trp repressor now we will look at this in vivo for an operon that is with the lac operon. - We’ve seen this before: it is a stereotypical operon with a promoter and an operator site as we’ve seen in previous examples. It has 3 different polysystronic coding sequences Z, Y and A that are going to be transcribed into a polysystronic message and then translated into 3 different proteins. These different proteins allow, in this instance E. Coli, to use lactose as a carbon source. - The proteins are listed in the slide. Slide 11 - The beta galactosidase hydrolyzes lactose – it breaks the beta 1-4 glucoside/ galactoside linkage that occurs between galactose and glucose so we have the beta 1- 4 galactoside linkage and that is cleaved by the beta galactosidase which gives rise to galactose and glucose which can each be used as downstream carbon sources metabolized further. - That was lac Z. Slide 12 - Lac Y encodes lac permease and this allows for transport of lactose into bacterial cells so when lactose is present in the medium, lac permease allows the cells to import it so that it can then be acted on by beta galactosidase to give rise to those utilizable C sources. Slide 13 - It turns out that the way in which the operon works is such that the concentration of lactose is what functions to alter transcription. - We can use the beta galactosidase activity as a measure of how much transcription is taking place at the lac operon. If you count the products of beta galactosidase per cell, we take a look at those per cell and in the presence of glucose, there is very low production of beta galactosidase. In the presence of glucose and lactose, there is a little bit of production but in the presence of lactose alone and the absence of glucose, you get a lot of production of the particular enzyme. - How does this operon function? Slide 14 - This is shown in the textbook as fig 7.39 and what it shows is that it involves important elements: the upstream promoter and a binding site that is located nearby to the operator, the binding of particular proteins (cAMP protein), binding of repressor protein. - What he wants to do is to break this down and see what’s occurring step by step. Slide 15 Binds to the operator - Lac repressor binds to the operator site – in doing so it prevents RNA polymerase from binding to the promoter. As a consequence only small amounts of lac mRNA are produced and we saw that there was a small amount of beta galactosidase activity even in the presence of glucose. So it is a bit leaky and that is important actually. Slide 16 Binds to the lac repressor - The presence of lactose, because there is a small amount of beta galactosidase produced, you have, first of all, the binding of lactose and the binding of a stereoisomer of lactose allolactose to the lac repressor. - Allolactose is a stereoisomer of lactose and it also binds to the lac repressor. Slide 17 Binds to the lac repressor - It is very important that even in the absence of lactose, there is a small amount of permease and a small amount of beta galactosidase, the permease allows import of even just a small amount of lactose into the cell. - The small import allows for the isomerization of the lactose into allolactose and the subsequent binding to the lac repressor which is no longer able to bind to the operator site. When this happens, the RNA polymerase can transcribe the lac operon but only very weakly. Slide 18 - There is another component and this is the CAP protein – the catabolite activator protein seen in the previous lecture and it contains stereotypical the helix turn helix motif that is able to bind to a given sequence in the DNA. - What he wants to do is to take a look at what happens to the catabolite activator protein and why it's called that and how it's functioning to regulate the lac operon. Slide 19 - We saw this slide already but what we haven’t looked at is simultaneously there is a molecule called cAMP that is produced in the absence of glucose due to catabolic processes. - The concentration of cAMP is contingent on whether catabolism has taken place or not so in the presence of glucose there are very low amounts of cAMP and in the presence of glucose and lactose again low amounts of cAMP made but in the presence of lactose alone, high quantities of this protein made. This is important because it influences how catabolite activator protein (CAP) functions. Slide 20 - Turns out that in addition to the operator site there is also a place where the CAP can bind and that is the CAP binding site indicated with a C. - Binding at the CAP requires the presence of cAMP. Without it, CAP can’t bind to the CAP binding site – so when glucose is absent, cAMP is produced as we saw in the lactose only conditions and as we can see here what occurs is as a consequence that CAP binds to the CAP binding site. RNA polymerase is then recruited and away it goes to generate the downstream components. - CAP facilitates RNA polymerase binding to the promoter and allows transcription to take place. So when glucose is absent, cAMP levels go up and transcription takes place. - By contrast when glucose is present, cAMP levels go down and we no longer have binding of CAP to the CAP binding site and low levels of transcription. Slide 21 - The lac repressor and the CAP working together. - In the presence of glucose and lactose what we have is no repressor binding but no cAMP present and as a consequence, no CAP binding. - In this point in time, the operon is effectively off but weakly on – very small transcription taking place. Slide 22 - In the presence of glucose and the absence of lactose the lac repressor is able to bind but no cAMP present so no CAP binding so the operon is truly in the off state. Slide 23 - When glucose and lactose are both absent we have repressor binding, cAMP present so the CAP is able to bind but because the repressor is bound, the RNA polymerase shown in grey is unable to do its job. The operon is said to be in the off state, truly off due to presence of repressor. Slide 24 - Finally in the absence of glucose but in the presence of lactose, we have an ideal situation. - cAMP is present so CAP is able to bind, allolactose is present so in this instance, the lac repressor is unable to bind & therefore RNA polymerase is able to be recruited. The operon is then on and away it goes doing its job transcribing the polysystronic message – what a great system
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