Wisconsin Public Television University Place Wednesday Nite at the Lab  Greg Richards, "Sugar Shock: How Bacteria Uses RNAs to Deal With Stress"  October 6, 2010 UP496   cc >> Tom Zinnen: Welcome, everybody, to Wednesday Nite at the Lab. I'm Tom Zinnen. I work here at the University of Wisconsin-Madison Biotechnology Center. I also work for UW-Extension. And on behalf of those, the Wisconsin Alumni Association, and Wisconsin Public Television, welcome to Wednesday Nite at the Lab. We do this every Wednesday night, 50 times a year. Tonight, I'm delighted to be able to introduce to you Greg Richards, who is from the University of Illinois. He'll be talking about "Sugar Shock: How Bacteria Use Small RNAs to Deal With Stress." Greg is originally from Sheboygan, Wisconsin.  Sheboygan is one of the great language centers of the planet. (laughter) >> And bratwurst centers. 

>> And bratwurst centers, absolutely. There's nothing "wurst" than bratwurst, except for the best. He went to the University of Wisconsin-Madison for his undergrad. He also got his PhD here in the Department of Microbial Science? >> Yep, in Bacteriology. >> In Bacteriology. And now, he's a post-doc at the University of Illinois, which is where I went for my masters degree, and by the way, for this group, that is where the phrase, "Wednesday Nite at the Lab" originated. That was a public science series done at the University of Illinois when I was a grad student there. So that's where the phrase comes from. I've had the great chance to talk with Greg on a couple of occasions. Three weeks ago, and now he drove up yesterday from Champaign. 

I think you're in for a really great treat tonight. Please join me in welcoming Greg Richards to Wednesday Nite at the Lab. (applause) >> Greg Richards: All right, thank you very much, Tom, for that introduction. Can you guys all hear me okay? Sounds good? All right, great. I'd really like to thank Tom and Nadia for the opportunity to come speak to you guys tonight. I'd also like to thank you guys for coming. I've heard a lot from both Tom and Nadia about you guys being a really fun audience. I'm really looking forward to getting to talk to you guys. And along those lines, feel free if you have questions throughout the talk to ask me. And of course, as usual, I'm happy to stick around after for as long as you guys want. 

If you're aren't sick of me yet, come on up and ask some more questions. So, yeah, as Tom mentioned, even though I'm coming to you from south of the border, in Illinois, I did my training here. So it's great to have an excuse to come back up here, especially with the gorgeous weather we're having. And along those lines, I thank you guys again for being here, indoors, when the weather's so nice in October. All right, so I'm going to talk to you guys today about stress, how organisms deal with stress, how they adapt to it. And in particular, I'm going to tell you about a type of molecule in cells called small RNAs. These are molecules that are required for the normal functioning of cells, in a variety of cells in different organisms. But in particular, they can often be involved in helping an organism adapt to different stresses in its environment. And in particular, I'm going to tell you about one example that we study in Illinois at the Vanderpool Lab, and that is in the bacterium E. coli, and it's a small RNA that helps it to deal with a particular stress. And that stress is a toxicity associated with sugar phosphates. 

And I'm going to get to telling you in a minute about that. All right. So, pretty much what I'm going to cover first is just talk a little in general about organisms and the kind of stresses they encounter in their environment. I'm going to give you guys a little bit of background on RNA, ribonucleic acid. I'm going to try to take you inside the bacterial cell, and kind of give you an idea of how these RNA molecules work to help the bacteria deal with stress. I'm going to finish up, again, and talk about some of the research we do in our lab, how E. coli uses a small RNA to deal with toxicity associated with sugar phosphates. And this toxicity has similarities to some of the cellular damage that's done in human cells during the course of diabetes. All right, of course, as I mentioned, all organisms have to deal with stress in their environment. There's a variety of different stresses that organisms encounter. Some of them, like being attacked by predators and having to evade them, are more immediate stresses to be dealt with than others, for example, getting a parking ticket. 

In addition, certain stresses, such as taking final exams, dealing with taxes, getting parking tickets, are more particular to one particular species. And others, such as dealing with cold, dealing with extreme heat, dealing with starvation, like this poor kitty here, are more common to a variety of different organisms. And certainly, bacteria are included in organisms that deal with this sort of stress. All right, so stress also occurs at the cellular level, both for organisms like bacteria that only consist of single cells, generally, and also other organisms, the cells that are within the organism. So, cells have to sense and respond to deal with a lot of the same stresses that larger organisms do, such as heat and cold, limitation of nutrients, different environmental stresses like osmotic pressure, radiation, different toxic pollutants in the environment. So, to be able to adapt to these types of stresses, an organism needs to be able to sense that the stress is there. Then it needs to make some change in the expression of its genes. And I'll get to what that means a little later in the talk. That results in a response to the stress that helps the cell to be able to deal with the stress. We're going to talk about an example of that. 

And as I mentioned, small RNAs are molecules in the cell that allow that cell to be able to respond to the stress in certain cases, and be able to survive a variety of stresses. >> Why are they called small RNAs? >> That's a good question. I'll get to that in just a second. I'm going to tell you a little more about the RNAs. But that is an excellent question. So, I'm going to get to that in just a second, actually. But before I tell you about that, I wanted to let you know that one of the reasons that we care about studying these molecules. And we found is that small RNAs are found throughout all domains of life, where they have been looked for. I'm showing you some examples here, as I mentioned, bacteria, certainly humans, as well as Bob Barker. I don't know if you consider him an actual human, but a sub-species, something like that? I don't know. 

But at any rate, I'm also showing you here a lot of representative kind of experimental models that are used in the lab to study small RNAs. Here, I'm showing you the worm, C. elegans. The fruit fly drosophila. Mice, zebra fish. Plants, certainly. And so, they're found widely throughout many different types of organisms. But what has been discovered is that, or rather, only in the past ten years has it been discovered how important these molecules are to the cell. All right, so now I'm going to give you, per your question, some background on what exactly RNA is. Are there any questions about the first part so far? All right, great. So, I'm going to tell you about RNA. 

And I'm calling it the other nucleic acid, because I think you guys are probably familiar with its more famous, publicity hogging, celebrity cousin, DNA. DNA is typically found, and you hear all about it in the news media, it's a really popular molecule. Well, poor RNA is also very important to the cell functioning.  Without RNA, the cell wouldn't be able to function. But you know, it's led a very bitter, publicity-free life, sitting off to the sidelines of its more famous cousin. So, I'm going to kind of tell you a little bit about what RNA does. Since I know a lot of you are familiar with DNA, I'm going to frame it in terms of comparing the two. So, they are both nucleic acids. One of the big differences between the two is that at a chemical level, they have a different sugar in their basic makeup. So, DNA actually, the sugar is deoxyribose, and in RNA, it's ribose. 

That's where the names come from. So for DNA, it's deoxyribonucleic acid. And for RNA, it's just ribonucleic acid. Probably a lot of you are familiar with the classic double helix structure of DNA, so it consists, typically, of two strands that are bound together. And RNA, although there are exceptions, is generally found as a single strand in the cell. So that's another difference between the two molecules. In addition, both DNA and RNA have four building blocks that are called nucleotides. They share three of the four in common, but RNA actually has one building block called uracil that's different than one of DNA, which is thymine. All right, so what actually makes a small regulatory RNA? Well, first of all, as you might expect from the name, they are small. So they are generally made of 50-300 of the building blocks of nucleic acids. 

And unlike most RNAs in the cell, small RNAs have a special function. They generally don't encode information like a lot of the RNAs in the cell. And I'll get to what that means more in just a minute. But the role of these RNAs in the cell are as regulators. So what they do is affect the expression of the genes in the cell. And it's often in response to some kind of environmental stress or signal. All right, so why do we study small RNAs? As I mentioned, they're found in all domains of life. And it's only in the last decade, however, that we've really begun to appreciate just how important these regulatory small RNAs are for the function of a variety of organisms. So here, I'm going to show you a history of discoveries that are related to small RNAs. Way back in 1874, a plucky young scientist named Friedrich Miescher isolated the first nucleic acid, which was actually DNA. 

And it wasn't till almost 80 years later that the structure of DNA was solved, and that it was proposed by some of the scientists that I'm showing you here. And it was shortly after that that Severo Ochoa showed how RNA is synthesized.  So, in the next year, and leading into the next decade of the '60s, a variety of scientists showed that there is an RNA molecule that is an intermediate synthesis of DNA to protein. And again, I'll get to what that means in just a minute. Then, also, in the early '60s, it was first proposed by Alexander Rich that RNA might have been the original genetic molecule in the origins of life, before DNA actually was. And following up on that, the hypothesis where several scientists, including Carl Woese, who I have to put a little plug in for the University of Illinois. He's there. So, what they discovered is that RNA isn't just able to transfer information, it can actually cause certain chemical reactions to occur. And then, this led to kind of more evidence that the earliest forms of life might have used RNA as a genetic material. So, that actually is what led Walter Gilbert to coin the phrase "RNA World." So, first of all, I have to say, isn't the guy like the most quintessential looking scientist you've ever seen? Like, if you picture a scientist in your mind, I just think of this kind of guy. 

So, he created this phrase "RNA world," and what it refers to is that it is thought that RNA was the original genetic material. And that is because not only can RNA help transfer genetic information, but it also has certain enzymatic properties that allow it to cause chemical reactions. So, it's thought that it might have supported the evolution of early life, even in the absence of DNA, which though it's great at retaining information, is rather chemically inert, or proteins, which are very chemically active in general, but they don't have the capacity for transferring genetic information from one generation to the next. And I'll kind of talk a little bit more about this in just a second. All right, it was also in the '60s actually that the first two small RNAs were identified in E. coli. Unfortunately, the Internet failed me as far as finding pictures of John Hindley and Beverly Griffin, who were the original discoverers of these molecules. But it wasn't until the early '90s that the first eukaryotic small RNAs were found. And that was in C. elegans in the worm model. That was Rosalind Lee, in the laboratory of Victor Ambros. But what the big take home point from my little history timeline here that I want to show you is that there's a really long time between the discovery of nucleic acid and really finding out about the importance of these small RNAs in the cell. 

And even similarly, from the first discovery of small RNAs in the cell, that was over 30 years ago. And it's really only in the last ten years that people have begun to appreciate what these molecules do in the cell. So here, I'm showing you some of the people who currently are working in the small RNA field, particularly in bacteria. And apart from, you can probably recognize the very obvious general trend of scientists getting more attractive over the course of time, based on the timeline I've shown you, you can also see that there are a lot more researchers working on this now. Here in the middle, I'm showing you the number of known small RNAs in E. coli over the years. And you can see, as I mentioned, in 1967, two were discovered. But you can see it wasn't until the years 1999-2000 that there was a significant leap in the number of known small RNAs that are important in the cell. And additionally, you can see over the course of the last decade, the number in the cell has just skyrocketed. The number of known small RNAs in the cell. So there really are a lot, just in E. coli alone, and they're a very important molecule. 

I keep telling you about them being important. But what do I actually mean by that? Well, small RNAs have roles in all sorts of types of cells. They have a lot of roles in just the normal functioning of various cells in humans. So, when scientists have looked for small RNAs in humans, what they found is that about a third of them are expressed in specific cells or tissues inside the human body. So it seems that there are likely to be certain small RNAs that are essential for both the differentiation of different tissues in the body, and also just for their normal functioning. So one example of this is embryonic stem cells. I'm showing you a picture of those here. In this case, it's been shown that certain small RNAs help keep embryonic stem cells in their undifferentiated state. In the case of germline or reproductive cells, it's been shown in mice egg cells, an example of which I'm showing you here, require certain small RNAs to be able to properly mature. So if you want to keep your reproductive line going and be able to transfer your genes to the next generation, small RNAs are absolutely essential in some cases for that. 

They're also essential for the normal development of muscle tissue during embryonic development. So in studies done in mice, what they found is that if certain RNAs are missing, this can lead to up to a 50% lethality of embryos. And the final example I'm giving you is that there are also many small RNAs in humans that have been found to be essential for the development of certain cells in the immune system. So, for example, formation of B and T cells, that keep your immune system functioning and healthy. And I'm showing an example of those here. All right, so small RNAs have also been found to be important in human disease. So, a big examples is many small RNAs have been found to be associated with cancer cells in one form or another. So, in some cases, certain small RNAs contribute to cancer cell formation, while other small RNAs have been found to actually suppress, to stop normal cells from being able to turn into cancer cells. So in this case, there are some that are harmful to the cell and some that are actually helpful. They've also been found to have important roles in heart disease. 

So some small RNAs have been shown they contribute to hypertrophy, which is a thickening of the heart walls, which is an indication of heart disease, and can also lead to complications, including heart attacks. So, again, similar to with cancer, some small RNAs have been found to contribute to this harmful trait, and others actually are involved in preventing it. Then, finally, another example. Certain viruses, such as Epstein-Barr, which I'm showing you here, and also Karposi's Sarcoma, have been found to utilize certain small RNAs to actually cause disease. This isn't just the case with viruses that infect people. There are also other organisms. So here, I'm showing you that small RNAs, in addition to having important roles in human health and disease, actually have the potential for use with disease treatment. So, there are a lot of active areas of research in this field. Not a lot of direct success yet, but a lot of explorations. So, the idea is that you can deliver small RNA to parts of the cell through injection, and then an example of that is being done in this photo. 

So, the goal in this case would be either to increase the presence of beneficial small RNAs, or prevent harmful small RNAs from being made. And this has potential applications to diseases such as muscular dystrophy, Alzheimer's, Parkinson's, and diabetes, and in addition, as I mentioned, heart disease and cancer. One of the big problems with using these as a potential treatment is figuring out how to effectively deliver a small RNA to the right cell. For example, again, with cancer, either being able to deliver a small RNA to a cancer cell that stops it from growing specifically, or get it to the right cells and prevent them from turning into cancerous cells in the first place. All right, so as I mentioned, there are other causes of disease in humans that also have important small RNAs that are required for their function. And a lot of bacterial pathogens have small RNAs that have been implicated in their ability to cause disease. I'm showing you a bunch of examples here. One is Brucellosis, which you get from infected meat or milk of cattle. E. coli. In E. coli strains that cause urinary tract infections, small RNAs have been found to be important for them to be able to cause disease. 

And as you can see, I have a list here of a variety of bacteria that cause important diseases. Everything from various causes of food poisoning to the cause of Cholera. I'm showing you here the cause of the Plague. And also, pseudomonas aeruginosa, which is the big cause of pneumonia in Cystic fibrosis patients. So, a lot of these bacteria require these things for disease. As an example, some species of Shigella have a particular small RNA that has not only been found to be required for their ability to cause disease, so if you think about it, if it didn't have this disease, it wouldn't be able to make people sick. So, this small RNA is very important. But it's also very important for the Shigella species to be able to acquire iron from their host. So, when they're growing in people, they are limited for a lot of different nutrients. A big, important nutrient that they're limited for is iron. 

And the small RNA actually helps  in their ability to acquire iron in the cell. All right, so I've kind of told you a little bit about what small RNAs are, and why they're important. But now, I kind of want to tell you what I mean when I say, you know, that they're causing something to happen in the cell. They're changing the gene expression. And that can help cells to be able to deal with stress, have a response to the stress and adapt to the stress, and be able to survive it. I'm going to try to take you inside the cell and give you a bit of an idea of what that means. All right, as I mentioned, cells can experience a variety of different stresses, just like we can. And they need to be able to sense that the stress is occurring, and then have some kind of change in gene expression. I'm going to try to give you an idea of what I mean by that in just a second. And then, that leads to a response to the stress to help them resist the stress. 

All right, I apologize, but in order to kind of tell you about those, I have to do a little bit of like a textbook introductory microbiology. So, hopefully, you guys won't get too bored by this. But I'll just plow ahead. And if you have any questions, just let me know. So, I'm going to kind of tell you again what I mean by a gene being expressed. We start with a gene, which consists of DNA. And in order to get that expressed, as you may have heard, DNA stores a lot of genetic information, but it can't directly do anything with that. You can kind of think of DNA as a blueprint for a building. So, the DNA needs to be transcribed into a type of RNA in the cell called messenger RNA. And it is named exactly for its function. 

It actually serves to transfer the message of DNA onto the next step. And it translates that message, and leads to a translation of that message into a protein. So, proteins, you can kind of think of DNA as the blueprint of the cell, the proteins are the actual parts of the building that have been added during the construction of the building. So, the proteins are kind of the workhorses of the cell. They have functions in the cell, a variety of functions. For example, they are involved in building parts of the cell. They're involved in transporting in nutrients and helping the cell to be able to use those nutrients as an energy source. All right, does anybody have any questions about that, or is that pretty clear, more or less? All right, great. Okay, so, there are molecules in the cell called regulators that can affect how much of the protein is made at a given time. So when I talk about changes in gene expression, that's what I'm talking about, how much either making more of something, if the regulation is positive, or making less of it if the regulation is negative. 

And as you can see from my high tech diagram here, the regulation can occur at any of these steps along the expression of the gene. So, some regulators can help more of the RNA to be made. Some of them can help more of the protein to be made, and some of them can help the protein to better do their function. And equivalent with negative regulators, making less of everything and ultimately having less of the function. All right, so how does this actually relate to what we're talking about with the response to stress in the cell? Well, when a cell experiences stress and senses that it's occurring, these regulators help change the expression of different genes in the cell. So, in the case of positive regulators, which are trying to activate these things, they actually will-- If you're responding to a stress, you generally want to make something, more of something that helps you to be able to deal with that stress. Whereas negative regulators, which generally repress the expression of genes. This is generally when you're trying to stop making something that is actually contributing to the stress, or is making the cell more stressed out. And so, in just a couple of minutes, get into a couple of examples of this. Yeah? >> Are all responses, in terms of the small RNAs, are they all responses to stress? >> Right, so that's a great question. 

They're not all responses to stress. So, some of the examples I showed you with human health, they're actually just required for just the normal functioning of the cell. But there are a lot of small RNAs that are important to dealing with stress additionally. That's what we study in the Vanderpool Lab. In just a minute, I'll kind of give you a couple more examples of that. Yeah? >> In the previous examples, which you just mentioned, not in responding to stress, is the small RNA still acting as a gene expression regulator? >> Yes, so that is-- Sorry, so he's asking, in the other examples, in human health and things like that, where small RNAs are involved in the function of the cell, are they also effecting the gene expression. And yes, that is correct. The role of small RNAs in the cell is to effect the expression of genes, whether they're involved in just the normal functioning of a cell, or if they're helping the cell adapt to different stresses. Yeah? >> How does this relate the epigenetic issue of methyl groups that also effect activation or repression? >> Right, yeah, so that's a good question. Small RNAs aren't directly related to that. 

But what you're talking about with methylation effecting expression is another way in which the cell can change expression, okay? It's not related directly to small RNAs, but it actually is another way that the cell can change that expression. >> So it's separate processes? >> Yeah, but they kind of work in a similar way to effect expression. Yep? >> So, small RNAs aren't like operands? >> Right, yeah, they're not like operands, that's correct. Yes? >> I want to follow up to my original question. If not all small RNA responses are to stress, what's the definition of stress and non-stress? >> That's a good question. I mean, we can kind of get into the semantics of what stress is. I'm kind of loosely defining it as, you know, the cell encountering something that is in some way harmful to it, or that it needs to make adjustments to in order to function properly. So, some small RNAs are just involved in kind of the normal, everyday functions of a cell, right? And what I'm talking about with stress is generally environmental stress, so things outside in the environment that are impacting its normal function maybe. And it needs to have a way to respond and deal with that. I'll kind of get to an example of that in just a couple of minutes. 

These are all really good questions. You guys are great. Yeah? >> You don't have to go into depth on this, but what is an operand? >> Oh, yeah, yeah. Okay, so an operand is where several genes in a row are kind of transcribed into the mRNA at the same time. And a lot of times, those genes will all have a similar function. So let's say you want to use some kind of sugar nutrients, or there might be several proteins that are required for you to break down that sugar and use it as energy. So that might all be encoded in the DNA, like right in a row in an operand. And then, they can all be turned on or turned off at the same time. >> So the functions, the different proteins these make, would tend to interact with each other? >> Well, they might. They may or may not interact. 

But they may be encoded in the same region of the genes. So they may interact in some cases, but in some cases, it might be a stepwise thing. Like, let's say you're breaking down a sugar. It might be broken down step by step, and it's kind of an assembly line thing. One protein might do the first step and then it passes that on to the next one. All right. So, when I talk about these regulators of expression, for decades it was thought that proteins were the main regulators of the expression in the cell, since as I mentioned, they're kind of the workhorses of the cell, they tend to be involved in a lot of the functioning. So what was found is that there were proteins that affect how a gene is transcribed in the RNA, and then how the RNA is turned into a protein, and even that can effect the functions of the proteins. So for a long time, it was only thought that proteins do this. But again in the last ten years, what we've been realizing is that these small RNAs in the cell have this similar role, and a lot of times are just as important as the proteins. 

All right, so the example that I'm going to tell you about today are actually small RNAs that bind to the RNA, as it's being translated into a protein. And as you might have picked up from the fact that it's generally single stranded in the cell, as opposed to DNA being double stranded, it has the capacity in some cases to bind to it's target RNAs through base pairing, similar to how the two strands of the DNA molecule bind together. I'll kind of go over an example of that in just a second. All right, are there any more questions on the general stuff? All right. Okay, so now I'm going to kind of tell you about what we study in the Vanderpool Lab at the University of Illinois, which is how E. coli deals with a particular stress, which is the accumulation of sugar phosphates. So first, why do we study this in E. coli? You know, there are so many interesting examples in human health and disease, what are we doing with messing around with this tiny, microscopic bacteria? Well, first of all, it's the undisputed number one lab rat of microbiology. It's been studied for decades. There's a lot known about it and there's a rich history of research that can kind of inform what we're working on and help us build on our discoveries. So, some examples of that are it's easy to grow in the lab, so in really fast times, it can have a doubling time of 15-20 minutes, which is a lot faster than raising mice or humans to try to do research on them. Plus, unlike humans, they don't get as grumpy if you try to subject them to stress. 

So that helps a lot. And also, apart from being easy to grow in the lab, we can manipulate the genes in the lab a lot easier than, for example, people. Again, we have one chromosome to deal with, as opposed to 46 in humans. So that's a lot easier also. But in addition, you probably all know that E. coli, as far as bacteria goes, is a pretty famous human pathogen, and it's a big cause of food poisoning in tainted meat supplies, and also on vegetables and things. It's also an important cause of bacterial infection of urinary tract infections. What you may or may not be familiar with is that E. coli actually also can be beneficial to humans. So, there's a very good chance that it's living in your intestine right now. It's not bothering you. It's a normal member of the intestinal microbes that live in people. 

It actually produces Vitamin K from scratch, so it can be an important source of that vitamin for you guys. So, not all the E. coli are bad. They can have kind of important beneficial and harmful roles in humans. All right, so then, another thing is why do we study this particular stress of sugar phosphate toxicity? Again, as I kind of mentioned, the sugar toxicity is associated with E. coli, and the cell damage that it causes has similarities to the sugar toxicity that's caused in diabetes. So this is kind of a general level. But there's potential for us in this simpler organism, studying it, to be able to apply this research to more complex organisms. All right, so what do I mean by sugar stress? You guys are probably actually probably more familiar with this than you think. You all know that sugar is a good thing in some cases, right? I mean, first of all, it's delicious. Second of all, it's an excellent source of nutrients and energy, right, as Willy Wonka taught us so wisely. But as with many things, too much of a good thing can be bad. 

And as you probably know, there can be bad effects to having too much sugar. So this is kind of roughly analogous to what's going on in the E. coli cell. So, what exactly is sugar phosphate stress? It is like the kind of general model I've been showing you. It's a stress that E. coli encounters. It needs to be able to change  it's gene expression in order to survive and respond to the stress. All right. Okay, so I'm going to kind of tell you about the research we do in the Vanderpool Lab. First, I kind of want to give you an idea of how the cell-- Before I get to what the sugar stress is, I kind of want to tell you how the cell normally brings sugar into it. So, there's a transport protein called ptsG. And that protein is responsible for bringing certain sugars into the cell. 

So if it senses sugars like glucose in the environment, it's like, great, I want to use that as a nutrient source, I'm going to bring it in. So it brings that into the cell. And it actually does that, it requires energy to be able to do that. And the energy in this case is in the form of transferring this phosphate molecule, breaking chemical bonds, transferring them. I'm not going to get into chemistry stuff. That gets a little boring, even for me. But the bottom line is, it's a form of energy that allows the transport of the sugar into the cell. So, when I talk about sugar phosphates, it's simply that to get the sugar into the cell, the transporter added a phosphate to it. So, in essence, it's kind of like an energy activated form of the sugar, which can then be used by the cell as a nutrient and energy source. All right, and that makes the cell very happy. 

But in other cases, if the transport of the glucose phosphate into the cell goes unchecked, it can be stressful. So the cell just starts to fill up with more and more of the sugar, and it just overwhelms the cell. And that makes the cell very sad, as you can imagine, impairing proper function of the cell, having this much sugar in the cell. All right, but thankfully, there's a response that allows the bacterium to deal with this, the glucose phosphate stress response. This involves, see this is all tying together now, isn't it? This involves the small RNA that is able to help the bacterium deal with it. So this small RNA has an excellent name. It's named SgrS, because it kind of looks like "sugars," right? But it stands for sugar transport related small RNA. And so, SgrS is a small RNA that helps to stop this glucose phosphate stress from happening. And it helps prevent glucose phosphate from coming into the cell. In addition, I told you that some of the regulators, or a lot of the regulators in the cell are also proteins. 

And there's a protein regulator that's also involved in this stress response. And the protein regulator is called SgrR, and it actually helps SgrS get made in the first place, and then SgrS can go on to kind of help deal with this stress. So these are kind of the major players in this response to this stress. All right, so I'm going to kind of show you how this stress response works. So here, I'm showing you, as I just did, the transport of the sugar into the cell. Here, I'm showing you the SgrS gene. So, SgrS isn't generally made at significant levels in the cell, unless there is stress occurring. I'm also showing you the ptsG mRNA to remind you that the ptsG also starts as a gene, and it gets transcribed into the RNA, and then that gets translated into this protein function. So, if the cell starts to sense that oh, there's too much sugar coming into the cell, I'm not liking how this feels, there's a signal. It's currently unknown. 

It's actually an area of active research in the lab. We don't know what the specific molecule is. But what it does, is it actually activates the SgrR regulator. So, what SgrR does, is it leads to the SgrS RNA being made once it's activated. All right, so the SgrS RNA, how it works, is that it's actually able to specifically bind to the ptsG RNA. And when it does that, it actually prevents that RNA from being made into new transporters. So, if you stop the synthesis of new transporters, you're not going to get any more of bringing this sugar into the cell, so you stop it from accumulating. You stop the glucose phosphate stress from happening. Yeah? >> How is the SgrS made from the DNA? Is it the same way the proteins are made? >> Yes, that's a good question. It's actually made, kind of in the same central dogma steps that I showed you. 

You start with the DNA, right? Then, generally what happens, if you're making a protein, it gets turned into the mRNA, and that's kind of the translator to make the protein. In this case, all we have to do, it's an RNA itself, right? So all it is, it's made from the DNA gene into an RNA, and then it just stops right there. So the RNA in that case is actually what's doing the work. That's a good question, though. Any other questions? Yeah? >> How are the small RNAs made? Are they encoded in the DNA? >> Yeah, exactly. So just like with other things in the cell, they're encoded in the DNA. So, with my little DNA here, I'm kind of trying to show you the gene, the SgrS DNA, right? And so, the SgrR protein helps that get transcribed from the DNA and turned into an RNA. So, yeah, that's essentially it. Yeah? >> How are they broken down? Are they broken down when the enzymes break down messenger RNA, or are they broken down separately by themselves? >> What do you mean? How is what broken down? >> Eventually, you have to figure out the signal once the job is done. So presumably, there are enzymes that break down the messenger RNA and also the SgrS. 

>> Oh, yes. That's a really good question. I can actually get to that just now. That's actually like I planted you to ask that question. That's my next slide. Okay, so the primary way in which SgrS works, is actually to stop the ptsG RNA from getting turned into the protein. But in addition to that, just in general, the cell is not happy to have double stranded RNA around. As I mentioned to you, generally in the cell, RNA is a single stranded thing. So, if the cell sees that this RNA is here, there are actually special protein enzymes in the cell that recognize this RNA. And they actually degrade it, which is what I'm showing you with the little PacMan here. 

They kind of chew it up and turn it back into the building blocks. That can be used to make other RNAs. >> So, specifically it finds the double stranded RNA and attacks that? >> Yes, there are signals sometimes in the molecules of the RNA, depending on which enzyme is degrading it. But that's a big warning sign for the cell in general that the double stranded RNA is there. These guys just don't like double stranded RNA, and they're going to chop it up. All right, any more questions about that? Okay, that's pretty much it. I hope that I've been able to show you again, as you know, all organisms experience stress. And in order to survive, they need to be able to adapt to that stress. And so, small RNAs are not the only molecule important for this. But they're important molecules in the cell that help organism cells to function in general. 

And they can also be involved in dealing with stress responses. So for example again, small RNAs such as SgrS can help organisms to deal with particular stresses. With that, I just want to thank everyone in the Vanderpool Lab, and all of these folks for funding, and all of you folks for listening. I'm happy to take any more questions. (applause)