Okay, there we go. Good afternoon. Just some reminders, the exam, exam two is next Monday, March 17th.
All students here, okay, I was not real pleased with how things worked out. I said I needed a room, double capacity, and everybody was in an individual seat, so heck with them. What it is, is there's, I don't know what's going on on campus, but there's a lot of, not very many large lecture classroom. So we're all going to be here. We're going to be friends.
Wear deodorant. That's all I got to say. So, and I'll be repeating that over and over because my fear is somebody's going to head all the way over there and then... and realize it's not there. There are 39 multiple choice or true, false, or multiple answer questions.
There will be one growth rate calculation question where I give you a table and say, calculate the growth rate. I will provide a formula for it, so you don't need to memorize that. There will be one energetics question. I will not be having you calculate Gibbs free energy. You can bring a calculator.
Please do bring a calculator. You will need it for the exam. unless you're really good with exponents and logs. But otherwise, you'll need it.
All right, any questions about that? Please keep... I've been really pleased.
People have actually been showing up for my APAS hours and asking questions, so I'm really pleased with that. All right. So we have started on regulation.
Again, just a reminder, this will not be in the exam. Right, so we're moving into new material. So some things. Regulation, first of all, needs to be specific.
So if you are regulating something, and these are general concepts in regulation, and you want to detect whether lactose is... is present in the environment and regulate genes based upon it, you can only regulate lactose catabolism genes. You're not going to regulate maltose catabolism genes or protein synthesis genes, genes involved in . in protein synthesis.
You're going to be specific to what you're sensing. Regulation also has to plug into the global state of the cell. If your cell is starving for amino acids and you cannot make proteins, it doesn't matter how much glucose is in the environment.
You're not going to degrade it because you don't have the proteins to do it. So the cell is going to react to that and shut off all transcription in cases where it's starving for amino acids or something. Regulation in bacteria is typically rapid, whereas if we took a regulatory pathway, for instance, let's say your response to a pathogen, that can take anywhere from four hours to seven days. Bacteria are going to respond to their environment in a matter of minutes.
You will see changes in transcription and translation in just a few minutes. And... full-blown expression of something in less than an hour. So it happens very rapidly, both on and off.
And much of regulation is tuned. What do I mean by that? If you've learned about regulation before, or when students first learn about regulation in this class, they often think that regulation is like a light switch. You either turn it on, or you turn it off. That's not how regulation works in the real world.
In the real world, you may have very little regulation. low expression of a set of genes, let's say in the absence of lactose, the lact genes will be very weakly expressed, but they will be expressed. Then when lactose is present, they may get turned way up, but in no case are they ever all the way off.
This is especially true in biosynthesis genes. So if you're talking about the synthesis of tryptophan, which is an amino acid that's used in proteins, you're not going to completely shut that off. And you're not going to express way more than you need because that's wasteful. So it's always tuned. It goes up and down depending on situations.
And you'll see that as we go through. So I have lots of examples of that. Okay, some general concepts that I want to talk about before we move into talking about specific operons.
Allosteric proteins. What's an allosteric protein? An allosteric protein is you have an active site on every protein.
That's where its substrate binds and is turned into product, right? Substrate binds. turns into product, right?
So active, binding here, product. Now, so here's the substrate binds, then there's the product. There's a second site where another molecule binds, and it's called the allosteric site. And this is a small molecule will bind to the allosteric site and change the enzyme's activity.
In the little cartoon I have here, our binding of the allosteric effect. to its allosteric site shuts off the enzyme. So the enzyme can no longer make its product, or the protein can no longer make its product in this case. There are lots of allosteric enzymes, and you'll see this, allosteric proteins I should say.
There's lots of allosteric proteins in regulation. Most often, these allosteric proteins will have their effect at the transcriptional level, like when RNA polymerase is making RNA, messenger RNA, or at the level of enzyme activity. And we'll talk examples about that.
Okay, there are numerous mechanisms of regulation. You could have a whole class. In fact, we do.
Microbio 470, just on regulation of gene expression in bacteria. I'm going to give you a sense, a taste of the kind of things that microorganisms can do, and we're going to talk about broad concepts, and then just focus on just a few operons that actually use those ideas. Now, there's numerous things you can do. You can change access to genes via DNA structure. This is actually something, you know, DNA arrangements and DNA methylation.
This is actually something that's done a lot in eukaryotes. Have you guys heard of it? of epigenetics.
Epigenetics is pretty amazing. It's something that happens in humans, where methylation of DNA will change the expression. And experiences that your mother had, when they were carrying you can affect your ability to express genes in your life downstream so it can have very interesting effects and they're still trying to figure that out that's very cool it's not something that happens in bacteria so I don't care we're not going to talk about it in this class but it's very interesting you can also have regulation at RNA right, and regulates whether RNA is made, initiation, elongation, and termination of transcription.
This is a major point of regulation in bacteria. That's why I have it in red. The ones in red are points that we will talk about and that are used quite often in bacteria. Another thing you can do is change the stability of the RNA. If you have an RNA that normally is stable for two minutes, And then it can be translated.
And all of a sudden, you change the stability to 30 minutes. You can get a lot more peptides translated with ribosomes because it lasts so much longer. It's another thing that you can do.
And then there's also proteins can bind to the RNA or small molecules and infect messenger RNA and infect its translation. You can also have regulation at the protein level. You can affect the translation and processing of the protein. That's more unusual.
We won't talk about that as much. You can also change a protein's stability. So you can make a protein less stable or unstable. So you can degrade it and get rid of it, and now you don't have that activity anymore.
Finally, protein function can be regulated by interactions with other proteins or small molecules or by covalent modification. We talked about small molecules when we talked about allosteric proteins. So.
Numerous mechanisms that you can regulate a DNA, RNA, and protein. The more common ones in bacteria are regulation of the start or regulation of transcription, the synthesis of messenger RNA, the stability of RNA, the stability of protein, and then regulating protein function by... Either other proteins bind to it, small molecules, or covalently modifying the proteins.
And again, we'll go through examples of this stuff. Okay, so I said one of the main regulations... is regulation of transcription. That's what we're gonna talk about now.
This is a very common method. Most often, the point of regulation is at transcription initiation. So you'll just stop RNA polymerase from transcribing the gene, making a messenger RNA. This usually involves a, uh-oh.
Okay, this usually involves a regulatory protein, and this protein will have two domains. One domain binds to the DNA, and here is a little diagram showing you what it does. It's, sorry, I got to learn to shut that off. Anyway, it will bind to the two grooves in the DNA right next to each other like this, and then that will block transcription. So fun little fact, these are identical domains.
So it's a dimer of the same protein. So let's call this protein X. There's two copies of protein X. And so if you look at the binding sites, the binding sites have to be the same because it's the same protein.
And if you go through the DNA, you can find these inverted repeats. And those inverted repeats, Right? We'll often suggest that's a binding site for a regulatory protein.
The second domain, so one domain binds to the DNA and does whatever it does. The second domain responds to some signal. And often, this is a small molecule.
So this is another type of allosteric protein. It has a second site that modulates the activity of this protein. And we'll go through examples of this as we go through.
These proteins will regulate elongation or termination or the initiation. And most of what we'll talk about is proteins regulating initiation. Okay, now we're going to start talking about these different ways of regulating.
And I'm going to turn the lights up a little bit because I'm going to use the board and I'm going to write these down as we talk about them. All right. The first one is called negative regulation. Negative regulation means turning something off, right?
And what that means is binding of the regulator, which is a protein, causes a decrease in transcription. So this is negative regulation, and this is transcription. So here is DNA bound to RNA polymerase.
Here's RNA polymerase binding to a promoter. And then downstream of this, you'll have... a sequence that your repressor binds to. In this case, it's the lac repressor, and you can see it binds it all up. And when it does that, this RNA polymerase cannot go through.
So binding of this lac repressor shuts off transcription. As you heard me say, the regulating protein is called a repressor. So if any regulatory circuit we are looking at, if I ask you if it's negative regulation, look for a repressor.
Or if a repressor is involved, it is negative regulation. All right, so that's negative regulation. Binding of the regulator turns off transcription or causes a decrease, I should say, and the regulating protein is called a repressor. Okay, now, negative regulation in practice, normally you have a really good promoter.
A lot of the diagrams are going to be like this one here. The black line represents the DNA, right, and this is how geneticists display genes, right? And then you'll have little boxes of sequence.
Big boxes like this are gene A, B, and C. These will be genes that encode proteins. Then you'll have other boxes. If I have a box with a P in it, that signifies a promoter, and that is where the sigma factor and RNA polymerase bind. Sigma factor recognizes the promoter, recruits RNA polymerase to it.
Then in some operons, you'll have what's called an operator sequence. That will be where the regulatory molecule binds. And in this case, we're talking about a repressor. So the repressor binds the operator.
RNA polymerase starts transcription at the promoter if it can bind to it. Okay, so normally you have a really good promoter. What does that mean?
It's something that RNA polymerase recognizes very easily. It matches the consensus sequence for that sigma factor very well. Sigma factor binds well, and boom, you get transcription.
So if the promoter's open, you get lots of transcription from that. from that transcript, that operon. Binding of the repressor, repressor shuts down, closes down, blocks the promoter, and RNA polymerase cannot transcribe the operon.
So that's negative regulation in practice. Okay, so if you inactivate the repressor, maybe by binding a molecule, so this molecule binds and it activates the repressor, it falls off the binding site, now the promoter is open. RNA polymerase can come in. Again, here's the gene, and it transcribes all the genes. So if the inactive repressor falls off the binding site, the corner's open, RNA polymerase can now proceed.
Okay. Now, we're going to talk about one type of negative regulation, and that is induction. So we're talking about negative regulation, and this is induction.
Okay, in induction, the repressor protein is active. The synthesized repressor is active without its signal molecule. Right? In induction, which is a type of negative regulation, The repressor protein is synthesized in an active state, and it doesn't need its signal molecule to be active.
It will bind to its operator, the operon, in the operon, and it will block transcription. Right? The repressor often responds to an inducer. Right?
So induction... Oops. The small molecule is equal to an inducer. The small molecule is an inducer. It's called an inducer.
Inducer binding the repressor causes it to fall off the DNA. And this is where students get confused. Okay.
It's negative regulation, but the presence of the inducer turns on the operon. It's called negative regulation because you have a repressor. That repressor binds to the operon and shuts it off, but the presence of the signal molecule, called an inducer, knocks the repressor off the operon and turns it on. All right, so induction is still negative regulation. That's why I've got it under this heading.
All right, the opposite. of induction is repression. Now, this is still negative regulation, but in this case, the synthesized repressor is inactive.
without its signal molecule. All right? The synthesized repressor is inactive without a signal molecule.
If it recognizes a co-repressor, which is the small molecule, the small molecule is called a co-repressor. it becomes active and it binds to the site. This is called repression. It's negative regulation because it has the regulatory protein, as they call it, shuts off transcription.
It's called a repressor. And it is turned on by a signal molecule, which is called the co-repressor. It binds to the site, and then transcription stops. Okay, so that's negative regulation. Let's look at an example.
And the example we're going to use is the regulation of the lac operon. So what is the lac operon? The lac operon is a few genes. It's lac Z, which is encodes beta-galactosidase. It's lac Y, which is a permease that transports...
lactose across the cytoplasmic membrane, and then it's galactoside acetyltransferase, which is a detoxifying enzyme. So here's lactose. Beta-galactosidase splits it into galactose and glucose, then those can go into glycolysis. So it's just one protein, one enzyme you need to actually run the pathway. All right?
Okay, so that's it. Now, interestingly, this will come in later. Back beta-galactosidase infrequently does a side reaction where it makes something called allolactose. Just remember that for now. All right, so in the absence of lactose, the lacI protein, which is the lac repressor, binds to the operator and shuts off transcription.
Okay? So there's no transcription when lactose isn't present. So there's absence of the inducing molecule. When the inducing molecule is present, it will bind to lacI and put it in an active state so they can no longer bind the operator.
By the way, these are reversible. So if the concentration of the signal molecule of the inducer decreases, it will go back to active, right? So it can switch between the states.
All right, now, if you go online, and this is why you don't trust Google, and you go look, and you Google lactose operon, and ask it how it works, it will tell you that the inducer is lactose. It's not. The inducer is actually the side reaction allolactose. So beta-galactosidase takes a little bit of this lactose.
And every once in a while, it converts it into allolactose. And this allolactose binds to the lac repressor and shuts it off. So let me repeat that again so that hopefully it makes sense.
Or I'll make it even more confusing. We'll see. In the absence of lactose, there's no lactose around, therefore there's no allolactose. LACI is synthesized in the active state. It binds to its operator and blocks transcription.
In the presence of lactose, Lactose is converted into allolactose. Allolactose is the inducer, right? The inducer, that's the small molecule that signals it.
It binds to lacI, it makes it inactive, and now transcription can proceed. Questions? Okay, that makes sense. Now, if you think about it, you go, wait a minute, how can lact-Z, which is beta-galactosidase, be present if the operon is turned off, right?
You need beta-galactosidase to turn on the operon because beta-galactosidase synthesizes allolactose. But if this operon is off, how can beta-galactosidase be there? Remember what I said at the beginning?
Operons are tuned. Expression is tuned. There's always a little bit of expression in the cell.
So there's a little bit of beta-galactosidase. And that little bit of beta-galactosidase can make some allolactose. That allolactose will then open up transcription, and off you go. Okay.
Let's see how we're doing. A deletion mutation in the lacZ gene. So a deletion mutation is where you completely remove the DNA of that gene. So therefore, absolutely, that enzyme is no longer going to be there. It's no longer going to be active.
A deletion mutation in the lacZ gene would do what? Give people a few more seconds to answer. Let's get everybody chiming in.
All right. I'm pleased. Okay, here's your responses. The microbe will be unable to grow on lactose. Almost everyone said that.
And that is the right answer. All right, woo-hoo. All right, come on, we got to show it.
Got to show it. Come on. What's wrong with you?
Okay, Top Hat decided to not do things. Okay, Top Hat doesn't want to show you, but that is the correct answer. So, nice job.
This is a smart class. Yes, question? I'm really tempted to answer that and yell at the person, but I won't.
All right. So, they're saying, please explain that I don't understand. Sure, I'd be happy to do that.
So, if we go back. And we look. This is the gene that we deleted.
LACZ encodes what enzyme? Beta-galactosidase. Yeah, you're saying it. Beta-galactosidase. Very good.
Encodes beta-galactosidase. What does beta-galactosidase do? Beta-galactosidase is the enzyme that splits lactose into glucose and galactose, right?
It also is the enzyme that makes allolactose. So you can't make... allolactose, right?
If you can't make allolactose, lacI is always going to be active, it's always going to sit on the operator, and you will never get transcription from that operon, or very rarely. All right, good. Okay, next question.
All right. I think it's being a weenie because here. Okay.
Let's go to lack. I don't know why it's being weird, but okay. I think I know why.
Hold on. I assign these as homework. All right. I'm going to unassign them. Now maybe it'll behave.
Okay, next question. The Lac Operon is an example of what? Right?
The repressor, you know, what is, is the repressor synthesized in an active state? And is the small molecule, what's it called? Okay, on this one...
We're a little mixed. So, everybody talk to your neighbors and see if you convince them what the right answer or the wrong answer is and then we'll re-poll. I'll give you like 30 seconds to convince your neighbor you're right. Okay, voting is open again.
Go ahead and change your vote if you want to. Okay, here are your responses. Right? We shifted more towards an induction, and that is the correct answer. Induction is indeed the correct answer.
Okay? No, for whatever, it's not highlighted. Induction is a credit answer. Why?
Well, first of all, this involves a repressor. Therefore, it is negative regulation. Okay, it cannot be positive regulation.
It cannot be fetal inhibition or attenuation because we haven't talked about those yet. All right? All right, so therefore, it's either going to be induction or repression.
The lac repressor is synthesized in an active state, and the molecule that binds to it, allolactose, is an inducer. So it is induction. So even though it uses a repressor, it's classified as induction. Okay, next question.
A mutation that activates the repressor lac-I would cause what? So you completely get rid of the repressor. Question. I just had a question going back to the first question where I asked about deletion of lac Z.
Is that only true for LACZ? Is it LACZ would inactivate it and make you unable to grow on lactose? What if you mutated LACY? What is LACY? LACY is the permease.
If you do, that's a great question by the way, if you inactivate the LACY, you can't transport lactose into the cell, you are still LAC-. You can't grow on lactose. What if you do the same thing to LACA in the laboratory?
It works just fine. The whole operon works just fine. What is lac A for then if you don't need it in the laboratory? Laboratory conditions are not environmental conditions.
It turns out lac A is needed to detoxify compounds that are often... found when lactose is present. And these are lactoses that have phosphates on them, right? And so you don't need to know this, but it turns out you need lackey in the environment to detoxify these toxic lactose byproducts, but you don't need it in the laboratory because we make the media. All right, so how did people answer this question?
Responses. Almost everybody said the microbe will synthesize the LAC enzyme all the time. That would seem like the right answer, but it's not.
All right? And I'll explain later why it's not. All right? The right answer is it has no effect or it depends on the media. All right.
But I will leave that mystery until later. Okay, so let's talk about positive regulation next. Okay, positive regulation is the binding of a protein termed an activator, not a repressor, and it increases transcription.
Normally with positive regulation you have promoters that aren't as good as in repression, so the promoter by itself can't really recruit RNA polymerase very well. It can kind of be recognized if RNA polymerase is given enough time to hang around with it, but it doesn't otherwise. What happens is the activator binds near the promoter, but doesn't block the promoter, and it recruits RNA polymerase. So there's protein-protein contact between the activator and RNA polymerase, and it kind of binds it to the DNA.
And then when RNA polymerase is there, it kind of looks around and goes, oh, there's a promoter, and off it goes. Sorry for the anthropomorphization, but hopefully it makes it more sense, or at least it's more fun. So then it then transcribes the DNA.
So an example of that is the maltose operon. The maltose operon has a bunch of MOL-E, F, and G, which are involved in degrading maltose. The MOL-T protein, the maltose activator protein's over here.
It's not shown in this slide. When MOL-T is around and maltose is present, maltose is its co-activator. MOL-T will then bind to the activator binding site. It then recruits RNA polymerase to here, and you get transcription. If maltose is not around, the mal-T protein, the maltose activator protein, will not bind to its activator site, and therefore you don't get transcription.
So you can see this kind of acts like the opposite of a repressor, right? When it binds, it recruits RNA polymerase and then causes transcription. That is positive regulation. So, let's summarize that.
Okay, the protein is an activator. If the small molecule binds, the activator binds. And if the activator binds, it recruits RNA polymerase.
And you get transcription. Okay? Alright, let's check your understanding of that. Hmm.
Okay, never mind. Actually, we'll just do this one this way. Somehow it got deleted out of my slide deck. Which feature of the multi-opera protein changes in the presence or absence of the co-activator?
Maltose. That's the question. I'll give you guys to think about it and I'll see if I can find it. Top hat's being weird today. Okay, it just got with the order somehow got switched.
There we go. Okay, which feature of the malty protein changes in the presence absence of the co-activator maltose? What is it? Okay, 19 seconds.
Okay. Most people said either the activity of the protein... Or the ability to protein bind DNA. And either one of those answers is acceptable.
So good job. Right? If the activity protein changes, then it's able to bind DNA. The site can bind DNA. So that's just fine.
Okay, so that's negative and positive regulation. We're now ELF transcription. Now we're going to move on to some other things that can change transcription and modify behavior.
And one of them is something really interesting called antisense RNA. Right? And in this case, here is the gene, right?
And you transcribe it, and you make a messenger RNA. And normally this would be translated into protein A. But sometimes there is this other gene that makes the complementary base pairs to a sequence in the messenger RNA. And this regulatory RNA binds to this. It is the complement to this RNA in a section, and it blocks translation.
This is called antisense RNA. If the transcript of the antisense gene comes directly from the complementary strand of the, let's say, gene A here, that is called cis-antisense RNA. If it's this kind of setup here, where gene X is somewhere else in the chromosome, here's the DNA. That is called trans, and I don't really care if you remember those terms, cis and trans, for this.
Just I want you to understand the concept of antisense RNA. You're actually transcribing a second section of regulatory antisense RNA that binds the messenger RNA and blocks its translation. That's antisense RNA.
And I thought I would give you a real-world example of this in... Right? The SimE protein.
A SimE protein is a protein that E. coli makes that will degrade all the messenger RNAs in the cell. Seems like a really stupid protein to make.
Oh, let's make something that wrecks everything in my cytoplasm. Alright? But there's a reason for it which I'll explain. SimR is transcribed from the same area as the antisense RNA that in almost all, you know, a lot of time will block Transcription or translation of SimE, right? So you make both of these.
Okay, so why is E. coli wasting all this time making SimE if it's also going to make SimR to block it? This is an emergency system that's only used if the SOS response is induced.
The SOS response is induced when you've been hit with something really bad, like a lot of radiation. And all that radiation has damaged all your messenger RNAs and maybe damaged your DNA and caused havoc. The last thing you want to do is try and translate these messed up messenger RNAs.
So in the presence of the SOS response, CIMR is inactivated and CIME is produced and then it degrades all the messenger RNA. All our messenger RNA is messed up because of this radiation. Now, Simi degrades it all.
It's an emergency system. But that's an example of antisense RNA. Make sense? Okay. Yes, question.
Yes, so the question is, wait, so is SIMR always on under the most normal conditions so that it blocks translation of SIME? Yes. It's only when the SOS response is induced that SIMR is degraded or inactivated, and SIME then can do its thing. Okay, so summary.
of transcriptional regulation. Negative regulation, the promoter is usually pretty good. The repressor blocks RNA polymerase access to the promoter and can be either induction or repression. Activation or positive regulation, the promoter is usually pretty poor.
An activator attracts RNA polymerase to the promoter. And there you go. And basically what you're doing is varying the accessibility of the DNA. Okay, I'm going to introduce one more topic here, and that is catabolite repression. Catabolite repression is an example of global regulation.
Right? Basic regulator, one regulator, one target. This is basic regulation. This is what we talked about for the LAC operon and the Maltose operon.
You have one regulator, one target. Global regulation is when you need to sense a bunch of things or regulate a bunch of genes based upon some response. The genes are often not adjacent on the chromosome, and these are called regulons.
There's all sorts of regulons and E. coli. These are just a bunch of examples. You do not need to remember one unless we talk about one specifically. But aerobic respiration, anaerobic respiration, catabolite repression, heat shock, nitrogen utilization, SOS response, etc.
And I will have more to say about that on Friday. Thank you.