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Lecture 10

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Biological Sciences
Dan Riggs

Lecture 10 – Gene Regulation I: Promotors and Control Circuits SPs: Figs 12-27, 28, 29, 32, 40, 44 Vocabulary:operon/regulatory gene/repressor/inducer/co-repressor/derepression/positive vs. negative control/cis vs. trans acting factors/deletion mutant/enhancer Figure 12-28: Structure of an Operon There are two distinct parts of an operon (shown in green). The first part is called the regulatory gene which encodes a repressor protein. The repressor protein governs the activity of the operon by interacting with the operator (O) sequence. The second part of the operon comes after the promoter (P) region and the operator sequence, where there are several genes in a row all involved in controlling the same biochemical pathway. When the regulatory gene is active, it makes a repressor protein which may or may not interact with the operator and depending on that interaction, it either helps to turn the gene on or turn the gene off. Now, let’s talk about the Lac Operon by talking about lactose metabolism. Lactose is a disaccharide made of galactose and glucose. When E. coli finds itself in the presence of lactose, and if the conditions are right, E. coli can use lactose as a carbon source for cell metabolism. Lactose in this situation is going to be referred to as the producer. When it is induced, a couple of gene products are made and one of them one very important product is called beta- galactosidase. Beta-galactosidase is an enzyme that recognizes lactose and cleaves it into its sugars - glucose and galactose. Glucose is the favourite carbon source. Early experiments on conduction of the LAC operon and E. coli look something like the graph on the right. On the bottom (x-axis) is time and along the y-axis is amount of beta-galactosidase enzyme or the amount of the messenger RNA. There is an established bacterial culture and at about four minutes, you add the inducer (lactose) and see what happens. If you measure beta-galactosidase messenger RNA (blue line) very shortly after you add galactose, the messenger RNA level goes up dramatically and it stays up until the inducer (lactose) is eliminated (for example if the culture uses up all the lactose or if the media is washed and removes the lactose). When the inducer is removed, you see is that the messenger RNA level drops very dramatically. Another thing you can see is that very soon after adding the lactose, the messenger RNA goes up (blue line), and the beta-galactosidase, which is the enzyme translated from the messenger RNA, goes up almost as fast as the messenger RNA does (red line). One of the reasons for this is that bacteria don't have compartments: there is a chromosome, RNA polymerase comes along and makes a message and as the message is being made, it is in the same compartment as the ribosomes so the ribosomes begin to associate immediately and translation begins sometimes even before the message is complete. That is the kinetics of beta- galactosidase, messenger RNA and enzyme induction. Inducible Operon: The Lac Operon The Lac operon is an example of an inducible operon. In figure 12 – 29, there is a red molecule in the middle which is an active Lac repressor molecule. When lactose is present, it binds to the repressor and changes its structure so now the repressor bounces off the operator; the structure has changed such that it cannot bind to the operator region. The role of the repressor is to repress transcription so if lactose is present the repressor is inactivated. Now the operon can be induced. RNA polymerize has a place to load, there is no roadblock in the way without the repressor being there, so transcription is going to take place. This is referred to as induction. (Remember that lactose is the inducer and this is the induction step). In the induced state, RNA polymerase is rolled off the messenger RNA and the ribosomes begin to associate. The translation of the messenger RNA yields three enzymes that are busy converting the inducer (the lactose) into the two sugars, glucose (as a carbon source) and galactose. After you finish eating your preferred carbon source and finish metabolizing the lactose, the supply of lactose becomes lower and lower. Most molecular interactions of transcription factors from DNA or small molecules of proteins are transient interactions so by lowering the lactose concentration, eventually the repressor is going to become active and return to its repressive state. It will change its confirmation such that it can bind to the operator. This serves as a roadblock for RNA polymerase. RNA can polymerase cannot work because there is something physically in the way of it getting to the structural genes. This is referred to as repression. Transcription is blocked. Repressible Operon: TRP Operon Figure 12-29 “Repressible” give the idea that it can be repressed or turned off (true) and also that it's default is on (true). The TRP operon is used to make one of the 20 amino acids called tryptophan. The biochemical logic is that tryptophan is needed for incorporation of proteins, etc. and we need a ready supply but if you don't have a lot of tryptophan around why bother to transcribe the operon and translate five different genes to make five different proteins to make something that you already have? The availability of tryptophan governs whether the operon is on or off. If tryptophan is present then you don't need to make more so repression takes place. That works like this: instead of the repressor being made active in its native form, it is made in an inactive state. The repressor has a structure that will not bind to the operator unless tryptophan is present. Tryptophan interacts with the repressor to change its structure and allow it to bind to the operator. In this case tryptophan acts as a corepressor. This then promotes the repressed state. If RNA polymerase is around it tries to bind to the promoter however there's a roadblock in the way. Now what happens. As tryptophan becomes used up by being incorporated into proteins etc. the concentration of the tryptophan falls. Remember that tryptophan is a corepressor bound repressor. As the level of tryptophan falls, that corepressor is going to lose its tryptophan and now you have an inactive repressor that exist and cannot bind to the operator. Now if RNA polymerize is around, it can bind to the promoter and with no roadblock in place it can conduct transcription and de-repression occurs. De-repression is a double negative so it can also be defined as reactivation. In the derepressed or activated state, RNA polymerase does its job and makes the RNA. Ribosomes translate the RNA and five different enzymes are made which can convert precursors to the end product tryptophan. In organic chemistry, the words Cis and Trans refer to substituent groups on one side of a double bond or another. In molecular biology and genetics, this has a different meaning. Cis refer to Cis-acting sequences or sequences within the promoter gene. Trans refers to Trans-acting transcription factors which are soluble factors that can move around. (Trans means cross which means they can move around.) If you think about “your favorite gene”, there is a coding region for that gene and there is a promoter region at the front. Trans-acting factor is a soluble factor such as sigma factor or Tata box binding protein that float around until they come into contact with a sequence like a Cis-acting sequence. Tata box is an example of a Cis- acting sequence and either sigma factor in prokaryotes or TBP in eukaryotes is the Trans-acting transcription factor that recognizes that Cis-acting sequence. Gene control circuits are like any other electrical circuit. They can be positive control circuits that promote the activity of something like gene expression or they can be negative control circuits which can turn off control depending on the active form of Trans-acting factor (eg: In the Lac operon, the repressor had to interact with lactose and was inactivated whereas in the case of TRP operon, the TRP repressor needed to find something else in order for it to be active.) So it depends on the active form of the Trans- acting factor that might affect that as binding to the target sequence - sometimes it can't bind sometimes it can bind. Lets go back to the Lac operon and use positive and negative control circuits and see how regulation is fine tuned. Look at the biochemical logic for the Lac operon. Lactose is made of glucose and galactose. In the notes, glucose and lactose have been underlined in the first sentence to distinguish the carbon source being talked about and what effect it has on another carbon source. If glucose is available, why bother to spend the energy to make enzymes to catabolize or break down a different carbon source (i.e. you like the glucose best why should you spend the energy to make some enzymes to convert another carbon source to something you already have - which is wasteful). Let's assume you don't have your preferred carbon source but you also don't have the alternative carbon source (no lactose). If you don't have the lactose, why spend the energy to make enzymes to break it down into something that you want - it's not there. For the Lac operon, both positive and negative control circuits have to be in place in order for gene expression to occur properly. You know a little bit about negative control because that is based on the repressor but we don't know much about positive control. Remember that glucose is the preferred carbon source. When glucose level is low, a small molecule known as cyclic AMP correlates inversely i.e. when glucose is low cyclic AMP level is high and conversely when glucose levels are high, cyclic AMP levels are low. Somehow these are in opposite balance with one another. When cyclic AMP is present when glucose level is low cyclic AMP serves as a small molecule and binds to a transcription factor called CRP (or cap) and the complex then activates the Lac operon (it sponsors the loading of RNA polymerase). This may be a little complex so far so let’s look at a few situations. Imagine you have the possibility of having the preferred carbon source glucose or the alternative carbon source lactose, one or the other or neither. There are four distinct possibilities for these two sugars. Scenario 1: when lactose is high and glucose is high. What is the biochemical logic? You have the preferred carbon source so why go through the trouble to activate the Lac operon and convert lactose into something you already have? Here is how that works: if lactose is high, lactose (or allolactose which is a isomer of lactose) binds to a repressor, changes its structure so that it is inactive and cannot bind to the operon so there is negative control that has been overriden. What about positive control? Positive control is due to cyclic AMP and this protein called CRP in textbook but called CAP in this figure. If you have CAP, there is no cyclic amp present because glucose is high and cyclic amp is low, it cannot interact with a promoter region to load RNA polymerase. But RNA polymerase is around the chromosome and periodically it goes through this region and there is a low rate of transcription but for the most part, the Lac operon is blocked because you have your preferred carbon source. Scenario 2: Suppose that the cell has used up its supply of glucose so the preferred carbon source is going down but biochemically there is an alternative carbon source that is available. Now it makes sense of biochemically to turn on the operon so you can convert the lactose into glucose. Since lactose is high, you build up the repressor and that keeps the repressor from acting on the operator so there's no roadblock in place - overridden negative control. The fact that you have low glucose means that you have high cyclic AMP which is a factor that interacts with CAP and allows it to interact with the promoter, changes the structure of the promoter, and allows RNA polymerase to load very efficiently. So lots of RNA polymerize goes through, there is a higher rate of transcription, lots of proteins produced quickly convert lactose into the preferred carbon source glucose. Scenario 3: Suppose we have low lactose and high glucose. What is the biochemical logic? We have the preferred carbons source so turn off the Lac operon. Since lactose is low the repressor is not bound to it and therefore it has a confirmation that can bind to the operator and create a roadblock for RNA polymerase. Negative control is in place and positive control is not being observed. That is the gluce level is high and cyclic AMP is low so CAP can’t bind to cyclic AMP because it has such a low concentration it doesn't load to the promoter and it can't attract RNA polymerase very efficiently so the RNA polymerase that does get there is blocked by the operator and so there is a very low rate of transcription. Often because you have the preferred carbon source and the alternative carbon source is in low supply. Scenario 4: there is a serious problem. You don't have the preferred carbon source and you don't have the alternative carbon source. Biochemically what happens is since lactose is low the repressor can exert negative control by binding to the operator. Positive control is in place i.e. glucose levels are low so cAMP is present, binds to CAP, alters the structure of the promoter such as to enable RNA polymerase to load efficiently so it is ready to move forward if only the roadblock were not in place. So there is a very low rate of transcription because the repressor is blocking the road. Now we are going back to figure 12 – 32, showing the various levels of which gene regulation can be exerted. You know transcription has to take place first, we just talked about the initiation of transcription by control circuits and in eukaryotes, the
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