What's up Ninja Nerds? In this video today we are going to be talking about DNA transcription. Before we get started if you guys do like this video please hit that like button, comment down in the comment section and please subscribe. Also down in the description box we have links to our Facebook, Instagram, Patreon, all that stuff will be there. Alright Ningeners, let's get into it.
Alright Ningeners, so with DNA transcription we have to have a basic understanding of just the definition. What the heck is transcription? And it's really a simple thing.
It's just taking DNA, okay, double-stranded DNA within eukaryotic cells and even in prokaryotic cells, and converting that into RNA. So it's taking DNA and making RNA. That's all transcription is.
But in order for transcription to occur, in order for it to take place, we need two particular types of proteins or enzymes, if you will, to facilitate this process. And I want to talk about those real quick, because these are very important. Now, transcription can be kind of different, okay? And it's important to know the differences between prokaryotic cells, we'll consider bacteria in this case, and eukaryotic cells, human cells like me and you. In prokaryotic cells, there's a particular type of protein that is needed in order for transcription to take place.
What is that protein? So let's say that we take this DNA strand here right we have this DNA strand on this DNA strand we have these blue portions that I've highlighted here as a box with some lines in it this right here for right now I want you to know is what's called a promoter so this is called a promoter region now a promoter region is a particular nucleotide sequence within the DNA And what it does is it allows for particular proteins like RNA polymerases and transcription factors to bind onto the DNA and then start moving through the DNA, taking the DNA and making RNA. So that's the first thing you need to know is within the DNA. there's a particular nucleotide sequence we'll talk about a little bit later called a promoter region and that's the first thing that we need to identify.
Let's say that we take this particularly for prokaryotic cells. So prokaryotic cells, and we'll just say like a bacterial cell. Prokaryotic cells use a very particular type of enzyme.
What is that enzyme? It's called a RNA polymerase holoenzyme. So it's called an RNA, we're going to put pol, polymerase, holo.
enzyme. Now that's a lot of stuff. Let me explain what this is and I'll show you the structure of it, a basic structure of what the RNA polymerase holoenzyme is. It's made up of two things.
One of the components of this enzyme is called the core. enzyme. And the core enzyme for this RNA polymerase holoenzyme consists of multiple subunits that they just love to ask you on your USMLEs and other exams. And these are, they contain two alpha units, okay, two alpha chains, proteins. It contains two beta units.
Technically, we say beta and beta prime if you really want to be specific. And then one more, which is called an omega unit, okay? So these are the primary components of the core enzyme, which makes up RNA polymerase.
What's important to remember is that these are what are going to really read the DNA and make RNA. That portion of the enzyme reads the DNA and makes RNA. The next The next component of the RNA polymerase holoenzyme is the portion that we need in order to bind to the DNA to the promoter region. Without it, we won't be able to allow for this RNA polymerase to bind to the DNA and transcribe it. This is called the sigma- right, or you can represent it like this, subunit or factor, if you will, okay?
These two components, the core enzyme, which is made up of the 2-alpha, the beta and the beta prime, and the omega subunit, as well as the sigma subunit, make up the entire RNA polymerase. Now, let me show you for example here. Let's say I represent the core enzyme as just this kind of blue circle with lines in it, and then we'll represent the sigma subunit as kind of like a pink circle with some lines in it, right? So let's imagine here. we have that core enzyme, which we're going to represent like this, and then the other component of it, which is the sigma subunit, which we'll represent like this.
That sigma subunit will then bind to the promoter region. Once it binds to the promoter region, then this core enzyme of the RNA polymerase will then release away from the sigma subunit, and it'll start moving down this DNA, and as it moves down the DNA, DNA will read the DNA from three to five and synthesize an RNA strand from that, which we'll talk about more detail later, from five to three. So it'll read the DNA and make RNA.
This RNA that we make from in prokaryotic cells with the RNA polymerase holoenzyme is very different from eukaryotic cells. In prokaryotic cells, that mRNA that we made from this one RNA polymerase holoenzyme can make all the RNA we need, whether that be our RNA within the prokaryotic cell, whether that be mRNA within the prokaryotic cell, or tRNA. within the prokaryotic cells. So that's very important. Big thing I really need you guys to take away from that is prokaryotic cells, they use one RNA polymerase which is called a holoenzyme made up of two components, a core enzyme made of these subunits and a sigma subunit.
The core is what reads the DNA and makes the RNA. The sigma subunit is what binds the RNA polymerase to the promoter region enabling it to transcribe the DNA. And whenever you make RNA within a prokaryotic cell from this RNA polymerase, it makes all the RNAs within that prokaryotic cell. In eukaryotic cells, it's a little bit different.
So let's talk about that. Let's say here we have three promoter regions that I want us to focus on. And this is all within eukaryotic.
cells. In eukaryotic cells, we need two different things in order to allow for transcription to occur. In this portion here, right, in this portion of prokaryotic cells, we only need one enzyme, which had two different components.
Within eukaryotic cells, each process requires a particular enzyme, an RNA polymerase, and a transcription factor. Let's kind of write that down. So let's say that we take this first promoter. We want to read this gene, this portion of the DNA, and make RNA.
And this is the RNA that we're actually going to synthesize right here, okay? From this gene. A particular enzyme, let's represent this in blue, since we've been kind of doing blue here. There's going to be a particular enzyme which is going to read this DNA, okay? And make this RNA.
There's a particular enzyme. What is that enzyme called? It's called RNA polymerase. But this is the.
the first promoter within the eukaryotic cells that we're talking about, right? So let's call it RNA polymerase 1. RNA polymerase 1 will read the DNA and make a particular type of RNA. But in order for it to do this, it needs a special protein that can bind to the promoter region, which allows for the RNA polymerase to bind to the DNA and read the DNA. What is that particular protein?
That protein, let's represent it here and let's do green. There's a particular protein which will bind here to the RNA polymerase and to the promoter and allow the RNA polymerase to bind to the DNA and start moving down, reading the DNA and making this RNA. What is this called? This is called a transcription factor, TF.
And there's many different types of transcription factors. The particular thing that I need you to remember for right now is that we call these transcription factors, which are utilized by RNA polymerases within eukaryotic cells, we call these general transcription factors. transcription factors.
We'll talk about very specific types with an RNA polymerase type 2 a little bit later but for right now two things I need in order for this RNA polymerase to be able to read the DNA and make this RNA. RNA polymerase 1 needs a general transcription factor to bind to the promoter, allowing the RNA polymerase 1 to then bind into the DNA, read it, and make RNA. What type of RNA does it make?
I have all the RNAs within prokaryotic cells from one RNA polymerase. But RNA polymerase 1 makes a very particular type of RNA, and this is called rRNA. Now, rRNA is very important because this is incorporated into what's called ribosomes.
Ribosomes. And ribosomes are utilized in the translation process where we take mRNA and from that make proteins. So we'll talk about this later in another video.
But for right now, first thing I need you to know is RNA polymerase 1, with transcription. factors, reads the DNA, and makes our RNA. Now that makes everything else pretty easy from this point.
Here's another promoter region of a particular sequence of DNA, right, within a eukaryotic cell. So this is the second promoter region. Another enzyme binds, another RNA polymerase, and not only just that one RNA polymerase, RNA polymerase here, but we also need a set of general transcription factors to bind to this promoter region. So general transcription factors we need to bind to the promoter region, enabling this RNA polymerase to bind to the DNA, read it, and then make what? Make these particular types of RNA we have here.
This is, well this was the first promoter, this is the second one, so we're gonna call this RNA polymerase 2. RNA polymerase 2 will bind to this promoter via the transcription factor, read the DNA, and make RNA. What kind of RNA is it going to be making? Big thing I need you to remember is it's making mRNA.
mRNA, you'll see later again, is the component. It'll have to go through some very specific modifications. that we'll talk about in great detail, and then eventually it'll be translated with the help of rRNA and another thing called tRNA at the ribosomes and making proteins, okay?
The other thing that you guys can remember, if you guys want to be scholarly or ninja nerdy, there's another RNA that's made here, and we'll talk about it a little bit later with what's called splicing, and these are called small nuclear RNAs, and these are involved in what's called splicing. And we'll get into that a little bit more in detail later, okay? But big thing, RNA polymerase 2 with the help of general transcription factors makes mRNA and SNRNAs.
RNA polymerase 1 with the help of general transcription factors makes RNAs. When the heck do you think this last promoter region of this sequence of DNA within this eukaryotic cell is going to make tRNAs? And it's the same process.
What do I need here? I need general transcription factors to bind to the promoter region. When that binds, that facilitates or it helps to allow the RNA polymerase type what?
Three to bind to the DNA and then read the DNA and then synthesize what? RNA. What type of RNA is it making?
The type of RNA that is being synthesized from RNA polymerase 3 is primarily tRNA, but a teensy little bit of snRNA. RNA is also made by RNA polymerase type 3. And if you guys really want to be extra ninja nerdy, technically even a teensy bit of our RNA is also made here as well. Okay? tRNA, what the heck does this do?
You'll see later that this is also involved in the translation process. It carries a particular amino acid and an anticodon, which is going to be involved in that process. And we'll talk about that in a separate video. So I know this was a lot of stuff to take away from this, but the big overall theme that I really just, out of all of this, what I want you to take away from this is this quick little thing here. That RNA polymerases, one, two, three.
Remember. R-M-T. RNA polymerase 1 primarily gives way to R-RNA. RNA polymerase 2 primarily gives way to M-RNA. And then RNA polymerase 3 primarily gives way to T-RNA.
These are the big things that I want you to take away from all of this. If you want to go the extra mile and be extra ninja nerdy, 2 and 3 also can give way to what? Small nuclear RNA. If you really want to go the extra mile, technically 3 can. can also give way to RNA.
But this is the basic thing to take away from what we just talked about. And then the other thing is, in prokaryotic cells, we don't need all of these. We need one RNA polymerase holoenzyme to make all the RNAs.
One last thing is, you notice in eukaryotic cells that we have particular transcription factors that are going to be needed for each RNA polymerase. The transcription factor in prokaryotes, technically, if you want to be specific, is the sigma subunit. because it's the portion that's binding to the promoter to allow the core enzyme of the RNA polymerase to read the DNA.
Okay, so that kind of covers the basic concepts of the two main things that we need in order for this transcription process to occur. Now, there's one other thing that I want to talk about very quickly before we really start talking about mRNA because that's going to be the primary topic here. I want to have a quick little discussion on how we can modulate the rate of transcription, either speeding it up or slowing it down.
Okay, so the... The next thing I want to talk about is very, very briefly on eukaryotic gene regulation. So I want to have a quick, quick, tiny little discussion on gene regulation. Okay. And the only reason I want to mention this is because this is very easy and it kind of makes sense along with what we're talking about.
But we're not going to talk about it in prokaryotic cells. We're primarily going to talk about this gene regulation. and eukaryotic cells.
We're going to have a separate video because it's more involved. We'll talk about gene regulation and prokaryotic cells with the lac operon and the tryptophan operon. We'll get into that. But in eukaryotic cells, there's two ways that we can modulate, and it's really easy. One way that we can modulate transcription is we have particular DNA sequences, particular sequences of DNA.
particularly palindromic sequences, which are called enhancers. And enhancers are basically DNA sequences. And the big thing I want you to take away from this, they can increase the transcription rate. So they increase the rate of transcription or the process of transcription. Okay.
And we'll talk about how they do that. The other thing that can regulate the transcription process or gene regulation in a way is something called silencers. And And silencers, they do what?
They decrease the transcription rate or the transcription process. Now, it's really straightforward. It's relatively simple. Let me explain what I mean. Let's say here we have a strip of DNA.
We're going to explain how this happens. So here's our strip of DNA. And remember this blue region?
What did we call this blue region that we talked about above? This was called our promoter region. And do you guys remember, let's take eukaryotic cells in this case.
What we need... in order for this process to occur. We needed a particular transcription factor to bind to that promoter region and then what else did we need in order for that to read the DNA and make RNA? You needed a particular RNA polymerase, right?
So we need an RNA polymerase depending on which one we're talking about, but depend on the type of RNA that we want to make and then a transcription factor. Okay? Now this is going to go read the DNA, this RNA polymerase will read read the DNA, and then make RNA, right? Now, here let's say that we have the promoter, and you can have this enhancer upstream from the promoter, or it could be down here, downstream, where we can't see it in this diagram, but it would be all the way down here. Regardless of where it is, it usually can be close to the promoter or it can be far to the promoter.
So you're probably asking the question, how the heck would an enhancer that's really far away influence a promoter that's all the way down here? How the heck does it do that? There's particular...
structures, there's different things that can activate enhancers and cause conformational changes of the DNA. And these are called specific transcription factors. You know why I really frustrate?
I got really deep into talking about specific and general transcription factors. The general transcription factors are what bind to the promoter region. Specific transcription factors, which we're going to really kind of do a different color.
Let's do purple. Specific transcription factors. Will bind to this enhancer region.
So this let's put specific transcription factors these will bind to the enhancer when they bind to the enhancer region It causes a looping of the DNA to where now the promoter was far downstream from this enhancer but when this specific transcription factor binds to the enhancer It causes the DNA to loop in a way that it's in very close proximity to the promoter region even though it's far upstream from it. And then what was bound to this promoter region here? Do you guys remember?
The general transcription factors and what else? The RNA polymerase. So now that these are in close proximity, guess what this specific transcription factor can do to this area over here?
It can act on these proteins and stimulate this reading of the DNA. The RNA polymerase is to read the DNA and to do what? Make. RNA Whether BMR and a rr and a tRNA so the whole point here is that Enhancers can be either far upstream or far downstream which makes it hard to interact with the promoter But if a specific transcription factor binds to that enhancer it creates a loop process bringing it in close proximity which can then stimulate the specific transcription factors and the RNA polymerases which are bound to the promoter to increase the transcription of RNA. What do you think silencers do?
The same process. We're not going to go into detail of it, but if you imagine I did the same thing, I put the silencer here and I have a specific transcription factor that bound here, it's going to fold it in a particular way bringing it close to the promoter, inhibiting that promoter region and slowing down the transcription process. It doesn't make sense, it's pretty cool too, right?
So I need you guys to ask yourself the questions because we're going to talk about these these general transcription factors. What in the world are the specific transcription factors. And I know that if you guys are the OG ninjas, you'll know these processes in and out.
You guys know when we make a protein, whenever we have like a cell signaling response, we've talked about this a million times here at Ninja Nerd, right? Let's say that we take a hormone like TSH, which stimulates thyroid hormone synthesis, right? TSH will act on a particular receptor.
We call these G protein coupled receptors, right? Like G stimulatory proteins. Those G-stimulatory proteins will activate something called cyclic AMP.
Cyclic AMP will then activate something called protein kinase A. Protein kinase A... Depending upon what type of transcription factor you need in this case, we're going to activate a very specific transcription factor for making what?
Thyroid hormone. So some type of thyroid hormone transcription factor that will be needed to bind to the enhancer, change the shape of it, activate the promoter, have the RNA polymerase read the gene that makes what hormone? Thyroid hormone.
And so you'd have this get read. You'd make an mRNA. That would.
would then get translated and make thyroid hormone. Doesn't that make sense how that process occurs? So we can increase the transcription and protein formation of thyroid hormone through this process.
The same thing exists with steroid hormones. If I took, for example, testosterone. You guys know testosterone, right?
Testosterone does what? Testosterone will move across the cell membrane. It'll bind onto a intracellular receptor.
When testosterone binds onto the intracellular receptor, what will that intracellular receptor do? Bind to the enhancer. When it binds to the enhancer, it loops it, brings it close to the promoter, stimulates the transcription to make proteins within muscle so that you can get jacked. That's the whole process of how we increase transcription. And the same thing would happen if we wanted to...
to decrease it, just we would have some type of repressing transcription factor binding to the silencer that would inhibit the transcription process. So I think we have a pretty good idea now of the basic concepts of eukaryotic gene regulation. Now let's spend most of our time talking about the transcription, particularly of mRNA. All right, so when we talk about transcription, we've had a basic concept of it, right? That we need RNA polymerases and transcription factors to read the DNA and make the RNA.
But the real one that I want us to primarily focus on, which is primarily important with transcription of DNA, is mRNA. That was the real important one. Now that's in eukaryotic cells with utilizing the what?
RNA polymerase type 2. In prokaryotic cells, we would just be using the RNA polymerase holoenzyme. So what I want us to do is I want us to go through particularly, and we already have a basic concept of how this is going to work, but let's go into the stages of transcription, particularly for mRNA within within prokaryotic cells and eukaryotic cells. The first stage that is involved here is called initiation of transcription. So the first step. that we have to talk about is called initiation of transcription.
Now this is the part that we've pretty much already familiarized ourselves with. Now within this, let's have our two cells that we're going to do initiation with. We're going to have our prokaryotic cells here on this left side of the board, and then over here we're going to have the eukaryotic cells here on the right side of the board.
What I want us to do is to have kind of a comparison, a side-by-side comparison of the initiation process. The first thing that we need to know is we've talked a little bit about this already, but this blue region. What did we call this blue region here again? This blue region was called the promoter region.
Now the promoter region I told you is a particular kind of like nucleotide sequence that is very, very specific and allows transcription factors and RNA polymerases to bind to the DNA. It's kind of a signal, if you will. It's like, hey, here I am, come bind to me.
And prokaryotic cells, the promoter region has particular types of like names and just weird stuff that they can ask you in your exams. So in the prokaryotic cells, they call this the negative 35 region, which means from the start point at which the RNA polymerase starts reading the DNA and making RNA, if you go back 35 nucleotides, that's kind of the... Point at which the RNA polymerases will bind in Prokaryotic cells another one is called the negative 10 region, but they wanted to give this one a name So they called it the prib no box Just meaning that it's negative. It's 10 nucleotides away from that start of transcription, right?
And then the last one here is called the plus one Region which is also called the transcription start site So it's going to be pretty much the nucleotide at which you just read and start making the whole process of RNA So these are the regions that you guys need to remember within prokaryotic cells These are the kind of specific promoter regions and eukaryotic cells the promoter regions have particular nucleotide sequences that we need to be aware of. These are called the TATA box, which means that you would have thymine, adenine, thymine, adenine. That would be a particular recognition sequence within the promoter in eukaryotic cells.
Or CAT, C-A-A-T, so cytosine, adenine, adenine, thymine. And the last one is a GC box. So if there's a TATA box, a CAT box, or a GC box, These are identifying nucleotide areas at which the RNA polymerases, type 2, and transcription factors will bind to.
That is the important thing, okay? Now, the next thing here is the polymerases. The RNA polymerases within prokaryotic cells, it's just one.
It's RNA polymerase holoenzyme. Right? We already kind of talked about that with the core enzyme, 2-alpha, beta, beta-prime, omega, and then the sigma subunit. All of that's needed to bind to the promoter region. And eukaryotic cells is a little bit more, right?
We said that we needed two things. We needed an RNA polymerase. and what are we making here? Initiation, and we're going to say that we're trying to make what?
We're trying to make mRNA transcription. So we're doing transcription, but we're making mRNA. So what was the particular RNA polymerase? One, two, three, R, M. M is for the mRNA. So RNA polymerase type 2 is one of the things that I need.
The second thing that I need is the general transcription factors. And there's just so many. so many of these that I don't know how important and how specific we really need to go into these. I'll give you some of them but I just want you to know that there's so many different types of them.
The main one if you had to remember one specific out of the tons of them I want you to remember transcription factor 2D. This is the one that I really want you to remember and the reason why is this contains a structure called the TATA binding protein. So this transcription factor 2D has a particular protein portion which binds to the promoter region, the TATA box.
But there's many other region transcription factors. And you can remember these by transcription factor two, and there can be H, there can be E, there can be F, there can be A, there can be B. So there's tons of these dang things. So I don't know how important it really is to know that, but the main one I want you to remember is the transcription factor 2D.
All right, so these are the things that we need in order to for initiation to occur. So let's take for example we're going to have on one side eukaryotic enzymes will bind and on this side the prokaryotic will bind right. So let's say here we take for example we'll make this prokaryotic RNA polymerase, we'll make this one blue And we'll make the RNA polymerase over here for the eukaryotic cells, just for the heck of it, we'll make it orange. Okay, just so we can distinguish the difference between these.
So what will happen? This whole RNA polymerase holoenzyme will do what? Bind to the promoter what will allow it to bind what subunit of it the Sigma subunit And if you really wanted to go back you guys remember we made that Pink okay for the eukaryotic cells. What do we need?
we need the RNA polymerase type 2. We said we're going to represent that with orange. So here's going to be the RNA polymerase type 2 and then what else do we need? We need those general transcription factors.
There's a bunch of them but what's the particular one that I really want you to remember here? Transcription factor 2D which contains the TATA binding protein. So it binds to the TATA box which is a promoter region in the eukaryotic cells then allows the RNA polymerase 2-2 bind to the DNA. Now once the RNA polymerase is bound to the DNA it's gonna start moving down the DNA strands reading it and making RNA.
So we've now started the process of transcription. That's all that's happening here. The next step is that once we've bound had this RNA, so let's write these down here for the prokaryotic cell this would be the, we'll put RNA polymerase and we'll put H for the holoenzyme.
And for that one up here this is going to be RNA polymerase type 2. Right? Once this is bound and it's in the DNA it's going to start reading the DNA. As it reads the DNA it'll make mRNA. That process by which it does that is called elongation.
So let's write that down now. So the next step is elongation to make the mRNA. Now with any elongation a couple different things happens and this is thankfully the same in prokaryotic cells and eukaryotic cells. So thank the Lord for that right. So let's just say that we take either one of these RNA polymerases.
Let's just for the heck of it we'll say Here's your RNA polymerase 2. Okay, here's your RNA polymerase 2 and it's reading the DNA. The DNA we already know has two strands. We're gonna call this top strand here, this top strand, son of a gun, this top strand up here we're going to call this the template strand. So the template strand. Also sometimes referred to as the antisense strand.
This strand down here we're going to call the coding strand. Now when RNA polymerases read DNA the strand that they read is the template strand or the antisense strand. So that's the first thing I really need you guys to remember is that the RNA polymerases, what strand do they read? They read, we're gonna put the template strand or also referred to as what else? the antisense strand and that's the strand that they use to make the mRNA.
They do not use the coding strand. So let's kind of put a little asterisk here that this is the strand that we're going to read. Now when it reads it, it does it in a way that you guys, if you guys watch our DNA replication video, this should be so darn easy.
Let's say here this end of the DNA is the 3 prime end. That means that this end is the 5 prime end. And remember, one strand of DNA on this side should have a complementary anti-parallel strand on the other side, which means that this is the 3 end on here, this has to be the 5 end on this side, and this has to be the 3 end on that side. What happens is this RNA polymerase, when it... and it binds into the DNA.
It does something very interesting. It binds to the DNA through the initiation process and then opens up the DNA. Who opened up the DNA before?
It was that whole, in replication, it was that whole replication complex. RNA polymerase does that. So the first thing we need to know is that RNA polymerase does what?
It opens the DNA. Now in replication, what else happened? You opened the DNA and you had those single-stranded binding proteins which kept it stable and kept it open, right?
RNA polymerase does that. on its own. So it also stabilizes the single stranded DNA molecules, right?
So it stabilizes the single strands. Then what was the enzyme and replication that opened up that unwound the DNA? Helicase.
Aronic polymerase has its intrinsic helicase activity so it also unwinds the DNA. After it unwinds the DNA then it starts reading the DNA. So let's say here as it reads the DNA DNA in this direction, three to five, it'll make mRNA that'll be going in the opposite direction. So it's going to read this three all the way to the five direction. And as it does that, it starts synthesizing mRNA, right?
And this mRNA will be synthesized in what direction? What will this be? This starts point? The 5 end.
And what would be this point? The 3 end. So we know the next thing that the RNA polymerase does, whether it be in prokaryotic cells or eukaryotic cells, is it reads the DNA from 3 to 5. Then it synthesizes RNA.
From what direction guys? 5 to 3. Very, very important. The last thing that you guys should be asking is, okay Zach, you also said that in replication the DNA polymerases... read the DNA, and then if there was an accident or a mistake, they would proofread it and then cut out the nucleotide. What about RNA polymerases?
Do they do that as well? Because it looks like they've done everything that was similar in DNA replication. That's the one thing that's controversial. So the only thing that's kind of relatively controversial...
is is there a proofreading function? We don't really know. It's still subject to study.
So that's one thing to remember. If you want to compare this, the proofreading function is somewhat uncertain at this point in time. Alright, so we have an idea now.
We've read this DNA and we've made RNA. I know we talked about this a lot in DNA replication. We're talking about it here and sometimes it really can be Fusing when you're saying five and three and I don't I don't I don't freaking get what you're talking about Zach So I want to take a quick little second and explain what the heck I mean when I say it reads it from three To five and synthesizes it from five to three a diagram.
I really think will clear this up for you Let's take a second to understand what I mean by reading the DNA three to five and then Synthesizing at five to three. I think it's really important to understand that so let's say here We have this strand of DNA. So this is this is gonna be our DNA template, if you will. Okay, so this is our DNA template on this side, the blue one.
And then this is going to be the RNA that we're going to synthesize utilizing the RNA polymerase type 2 and eukaryotes are the RNA polymerase holoenzyme and prokaryotes. Now, when we're making this RNA we have to read the DNA in what direction? The 3-end to the 5-end.
What is the 3-end? You guys remember the video on DNA structure? It's the OH. So this is going to be the 3-end.
What's the 5-end? It's the phosphate group. So the phosphate group is going to be the 5-end.
So I have to read this starting at the OH portion towards the 5-end where the phosphate is. So the RNA polymerase, let's pretend I'm the RNA polymerase. I'm walking by, do-do-do-do-do-do-do.
I find the 3'man. I'm like, oh, there it is. Okay, I'm going to move up.
Oh, I found the 3'5'. Let me just feel this up. Oh, I feel my nitrogenous base.
The nitrogenous base that it feels. is adenine. So it picks into its little satchel of nucleotides. It's like, okay, this is adenine.
The complementary base for it is thymine. Uh-oh, no, that's not correct. Because you guys know that if we're taking DNA and making RNA, what's the one nucleotide that switches from DNA and RNA? Adenine is no longer complementary to thymine and the RNA. It is uracil.
So the RNA polymerase will come, read, find the three end, read the nucleotide, and say, oh, okay, this is an adenine, reach into its satchel of a bunch of nucleotides, and pull out uracil. When it pulls that out, it then puts the... nucleotide in a particular orientation. What's the orientation? We said it reads it from three to five and synthesizes it from five to three.
What's the five end? Here's the nucleotide. The five end is this phosphate group. The three end is is this OH group.
So it's going to kind of flip the nucleotide the opposite direction and make sure that the nitrogenous base here is what? Uracil. Then when it does that it's going to go to the next one so it's going to continue it's going to go to the next point.
Here's where the next OH group would be right the three prime end. Reads it finds that finds the nucleotide and says oh the nitrogenous base here is T. Let me reach into my satchel of a bunch of different good old nucleotides. I'm going to read it. T goes with A.
I'm going to put my nucleotide and I'm going to flip it where it's 5 prime end going down, 3 prime end pointing up. And then the nitrogenous base which is complementary to the T is A. When it does that, it then fuses the 3 prime end and the 5 prime end together, making a bond. What is that bond called?
The phosphodiester bond. And the same process occurs. So then it'll do what? Let's fix this 3 prime end. prime in there.
It'll then go, go to the next nucleotide. Here's the three prime in where the OH group is. Reads it, finds the nucleotide, says that it's a G, reaches into its satchel, pulls out a nucleotide with the cytosine. When it does it, it flips it to where the five end is on this side. There's my phosphate.
The three prime end is upwards. And it says, oh, the nucleotide that goes with this is with the nitrogenous base C. Then it says, oh, I have my five prime end situated close to the three prime end of the preceding nucleotide. let me fuse these two together and make my phosphodiester bond. And just for the heck of it because repetition I guess is helpful, we go reads this says okay next one here's my three prime in where the OH group is, read it, find the nitrogenous base, it's a cytosine.
digs into its satchel, pulls out the nucleotide, guanosine, I'm sorry, the guanine nitrogenous base. Then when it does that, it situates it where the 5'end is situated down, 3'end is situated upwards in this case, and then the nitrogenous base is guanine. Then it says, oh, my 5'end, I can stitch it to the 3'end of the preceding nucleotide and form my phosphodester bond. And that's how we make RNA, reading it 3 to 5 and synthesizing it from 5 to 3. Dang, we good.
All right. Now that we've done that, the last thing I need you to understand is that RNA polymerase is a very important enzyme within eukaryotic and prokaryotic cells. A question that can come up, and it's so dumb and annoying, but you should know. know it is that In eukaryotic cells, we can inhibit the RNA polymerase by using a kind of toxin, a manitin.
It's for mushrooms. And this can inhibit the RNA polymerase within, we'll put, eukaryotic cells. Okay? There's another drug, which they love to ask in the exams as well, called rifampicin. It's an antibiotic.
And this inhibits the RNA polymerase within, if it's an antibiotic that's good against bacteria, prokaryotic cells. So this would inhibit the RNA polymerase within prokaryotic cells, which would inhibit what? The part of the...
initiation, the elongation, basically making RNA. If you can't make RNA, you can't make proteins. If you can't make proteins, you can't perform the general functions of the cell. So this is kind of from a poisonous mushroom, which is stupid to know that, but they like to ask it on your exams. And then rifampicin is an antibiotic, which they also love to ask.
Okay, now we've talked about elongation. We've made the dang RNA. RNA polymerase is working real hard. The last thing we've got to do is we've got to just end it.
We don't need any more RNA. We've made the RNA that we need to make the protein. That is called termination. All right, so we talked about elongation.
The next step, the last step, really, that we've got to discuss here is termination. We've got to end this whole transcription process. So the last step is termination. Now, unfortunately, Unfortunately, termination is probably one of the more annoying and complicated ones, unfortunately. And it is different in prokaryotes and eukaryotes.
That's why it kind of makes it a little bit frustrating. But termination is basically where we've already made our RNA transcript. And we just need to detach it or disassociate it away from the DNA and prevent that RNA polymerase from reading any more of the DNA and making any more RNA. So just stop transcription. How do we do that?
In prokaryotes, there's two mechanisms. One of the ways that this happens is through what's called row-dependent termination. So one is via this process called row-dependent termination. And it's really simple, believe it or not. So let's say here we take...
the prokaryotics. We picked blue for our RNA polymerase. So the RNA polymerase, here's our RNA polymerase.
It's reading this DNA. As it's reading the DNA again, what is it making from it? You guys remember it's making the... RNA in this case it could be any RNA it could be the mRNA tRNA RNA whatever As it does this there's a protein called Rho and what Rho does is this Rho protein will start moving up the mRNA and as it moves up the RNA that's being synthesized by the RNA polymerase as it gets to this RNA polymerase It basically says hey, it just punches the RNA polymerase off the DNA if If you punch the RNA polymerase off the DNA, is it going to be able to continue to keep breeding the DNA and making any more RNA?
No. So that terminates the transcription process. So the big thing I need you guys to know here is that with the Rho-dependent termination is Rho protein.
causes RNA polymerase to break away, to disassociate if you will, okay, to break away from the DNA. Okay? Alright, beautiful. The next mechanism within prokaryotes is row independent termination.
So we don't use the row protein. So we call this row independent termination. Now with this process it's a little bit more complicated and a little annoying. Let's say here we have the DNA right and within the DNA we're gonna mark these here we're gonna say this is our template strand right so this strand is the template strand right or the antisense strand and then this is going to be our coding strand. So which one does the RNA RNA polymerase read?
It reads the template strand or the antisense strand. There's a particular like thing called inverted repeats that form within the DNA that the RNA polymerase is reading. So what happens is this RNA polymerase will bind to that template strand and it'll start reading it, making the RNA. As it starts making this RNA, it encounters a particular sequence of DNA called inverted repeats. Let's write these inverted repeats out in kind of a nice little color.
Let's do orange. And let's say here we have an inverted repeat where we have CCGG and then a bunch of nucleotides that we don't care about. And then here we'll have...
GGCC. Okay? Then we're just gonna have, this is the template, again, on the coding strain it would just be be the complementary base.
So if this was CC this would be GG, CC, we don't really care about these nucleotides, CC, GG. Right? The RNA polymerase is going to read this template strand.
What happens is, right, is you're going to get this kind of strand here where you'll have a bunch of nucleotides already kind of made up here and then it reaches this kind of like inverted repeat area. And what happens is it reads this and then basically everything you read within the template strand should be the same as it is in the coding strand because it's the complementary base. So you'll have GGCC that it'll make, a bunch of nucleotides we don't care about, and then CCGG.
What happens is whenever this RNA is kind of coming and being transcribed from the RNA polymerase, something interesting happens where some of these... C's and some of these G's on this portion have a strong affinity for some of the C's and some of the G's in this portion of the RNA. And as they start having this affinity, they start approaching and kind of wanting to interact with one another via these hydrogen bonds.
And so it creates this really interesting kind of like hairpin loop, if you will, where there's a bunch of G's and C's within this kind of hairpin loop. that are kind of interacting with one another. And what happens is that hairpin loop is what triggers the RNA polymerase to pretty much hop off of the DNA and terminate the transcription process.
Because what happens is once you form this hairpin loop, what will happen is there's going to be particular enzymes that will bind to that portion and cleave the DNA, the RNA away from the RNA polymerase. So the big thing I need you to note know within a row independent termination is that you'll hit this area the RNA polymerase will be transcribing reading the DNA making RNA it'll hit these areas of inverted repeats when these inverted repeats are made they create this thing called a hairpin loop This hairpin loop will then trigger particular cleavage enzymes to come and cleave a couple nucleotides after that hairpin loop to cleave that away from the RNA polymerase and then Here you have your RNA that you formed. So that is one of the ways that we have termination road independent via prokaryotes.
The last termination mechanism is going to be eukaryotic cells. Now how does this work? This one's actually relatively simple. So we had the RNA polymerase in eukaryotes and this was orange. It's binding to the DNA.
It's reading the DNA. As it's reading the DNA, it's making RNA. As it starts making this RNA, it hits a particular sequence where when it starts reading the DNA and makes RNA, it makes a particular sequence of AAUAA.
A. Okay, so what is the nucleotide sequence here? Let's write it out. This portion here will be AA, U, AAA. This is what's called a polyadenylation signal.
So what is this called here? This is called a polyadenylation signal. And once this kind of nucleotide sequence occurs, so it's kind of now that we know what that nucleotide sequence is, let's kind of just put like this. Here's that nucleotide sequence, that polyadenylation signal. signal that's been synthesized or formed by the RNA polymerase with the new karyotes.
Once that happens it activates particular enzymes and those enzymes will come to the area here and cleave the RNA away from the RNA polymerase separating out this RNA away from the DNA and the RNA polymerase. And then again what will I have at this portion here just as a kind of a diagram. my dramatic portion here, this will be my polyadenylation signal. And this is important because we're going to talk about post-transcriptional modification in a second.
So I know this was a lot of crap. Just really quickly recap because this is one of the toughest parts of transcription is termination. Prokaryotes, there's two ways, row dependent, row independent. With this one, you need a row protein to knock the RNA polymerase off. If you don't have him, he can't make any more RNA.
The other one is row independent. You don't have a row protein. The RNA polymerase is reading the DNA, making RNA, and it hits these areas of inverted repeats. These inverted repeats, when they're made within the RNA, it creates a hydrogen bond interaction between them, which causes it to loop, forming a hairpin loop. that signals particular enzymes to break the RNA away from the RNA polymerase, and we've made our RNA there.
The last one is in eukaryotes. The RNA polymerase... This is reading the DNA, and it reaches a particular sequence of nucleotides where it reads, and then makes AAU AAA. A polyadenylation signal which activates enzymes to come cleave the RNA away from the RNA polymerase, terminating the transcription process. That really hammers this home.
Let's now talk about post-transcriptional modification. We know at this point how to take DNA and make RNA, right? We talked about all the different types of RNA, utilizing the RNA polymerases, utilizing the transcription factors. We talked a little about gene regulation.
We even went through all the stages of transcription. Taking the DNA and making the mRNA all the way up until the point where we've finally made the mRNA and broken it away from the DNA. Unfortunately, that's not it for transcription. Now, we have this mRNA, right?
So, basically, what have we covered up to this point? We took the DNA. We read. Let's just say here.
At this portion, I'll just put here's our promoter. Our RNA polymerase has read this gene sequence. We hit a termination sequence. Let's say here's our termination sequence that we talked about here. And once we hit that termination, sequence, the RNA polymerase will fall off.
And then from this, you'll make the RNA. So this was pretty much the basic aspects of the transcription. But now we got to modify this.
Now here's the thing. It's actually kind of... of a misnomer to say that this is mRNA.
It's technically not mRNA right now. So this piece of RNA that we made, okay, and this process of post-transcription modification, this only occurs, it's very important, let me actually write this down, this only occurs in eukaryotic cells. So that's nice. All this stuff that we're going to talk about here is only in eukaryotic cells. It doesn't happen in prokaryotic cells.
So they just make their RNA and that's it. So technically, this immature mRNA, if you will, we actually give it a very specific name. We call it heterogeneous nuclear RNA.
Now, this heterogeneous nuclear RNA is kind of an immature mRNA that has to go through some modifications to really make mature mRNA that then can be translated to make proteins. What are those modifications? The first thing that we have to do...
is we have to put something on one of these ends. So now we got to know a little bit about the terminology of the ends of this immature mRNA or HNRNA. On this end, we're going to call this the 5'end. What's on that 5'end?
Do you guys remember? The phosphate groups. What's on this end?
The 3'end. What's on the 3'end? The OH group.
Okay? Now, something very interesting is on the 5'end. On the 5'end of this heterogeneous nuclear RNA, or the HNRNA, you have a triphosphate, which we're representing here with these orange circles.
An enzyme comes to the rescue and cleaves off one of those phosphate molecules. What is that enzyme called? It's this orange little cute enzyme. This orange enzyme is called RNA triphosphatase.
And what it does is, is it comes and cleaves off what portion? It cleaves off one of these phosphate groups. It's going to cleave off one of the phosphate groups. So now I only have two phosphates on the end of this 5'end. Then another enzyme comes in.
And it says, hey, there's only two phosphates here. I can now add something on here. And I'm going to add on what's called a GMP molecule. What am I going to add on again? I'm going to add a GMP molecule, which is guanosine monophosphate.
So we're going to represent that here, which we add on the phosphate for the guanosine monophosphate, and then we're going to just represent this as the guanosine. So this is our guanosine, and that blue circle there is the phosphate on the guanosine. So what does he add on?
Technically, he adds on to this little two phosphates. Right? It adds in GTP, but when it does that, two phosphates are released in the form of pyrophosphate, which then get broken down by pyrophosphatase into individual phosphates.
So if I took GTP and I removed two phosphates, what am I left with? GMP. So it adds on this GMP group onto that 2-phosphate end on the 5'end. So this enzyme that adds that GMP on in the form of GTP is called guanylyl transferase. Guanylyl transferase.
Beautiful. So this last enzyme here which is involved in this step here on the 5'end is going to add on a methyl group onto one of the components of the guanosine monophosphate. It's actually like one of the seventh components on that structure.
It adds on a methyl group and so at the end of this, this enzyme which adds a methyl group on, what do you think it's called? Methyltransferase. At the end of this process, where you took the 5'-man, which had three phosphates, got rid of one, took the guanolat transferase, added on the GMP, took the methyltransferase, added on that methyl group, you formed this complex here, and we call this whole complex that we just...
added on a 7-methylguanosine group. Okay? And that's on that 5 prime end.
This is called capping. This is called capping. So whatever we've just done on that 5 prime end, we're on this five prime end is called capping. What the heck do we do all this stuff for? The whole purpose of capping is to help to initiate translation.
So this sequence, this kind of five prime end with that seven methyl guanosine or that five prime capping, if you will, it's kind of a signal sequence, if you will, that allow for it to interact with the ribosome and undergo translation. The other thing it does is it it prevents degradation by nuclease enzymes that want to come and break down the RNA. So it helps to prevent degradation, helps to initiate the translation process.
One more thing that they, it's a dumb thing to know, but they love to ask it, is that there is a particular molecule that this methyl transferase uses to add that methyl group on. And sometimes it's really important to know it. And this is called S-adenosyl. methionine also known as SAM. SAM carries a methyl group it's like a methyl donor if you will.
It gives that methyl group to the methyl transphrase and the methyl transphrase adds that methyl group onto the guanosine monophosphate forming the 7-methylguanosine or that 5 prime cap. Okay so that's the first thing that happens. Now we got to talk about the 3 prime end.
On the 3 prime end we had that OH group right that's the OH end but do you remember and eukaryote, there was a particular signal that generated, that terminated transcription. What was that nucleotide signal? Do you guys remember?
Test your knowledge, guys. AAUAAA, right? That was that polyadenylation signal.
Do you guys remember that? The polyadenylation signal we talked about in eukaryotes? That polyadenylation signal is recognizable by this cute little purple enzyme here.
This cute little purple enzyme is called poly-A polymerase. It's called poly-A polymerase. What it does is on this hand, it has a bunch of adenine. nucleotides, right?
So it contains a lot of nucleotides containing the adenine nitrogenous base. It takes one end and identifies that polyadenylation signal, takes the other end and adds on all of those. adenine nucleotides, a bunch of them, sometimes up to 200 adenine nucleotides.
When it does that, this forms a tail on that three prime end with a bunch of adenine nucleotides, and we call this the poly a tail. So the poly a tail, what's the purpose of this? It's the exact same thing.
Helps to initiate the translation process. and helps to decrease degradation by what kind of enzymes? Nucleases that will try to come and break down that end. Okay, the other thing that they do is they help transport this HN RNA.
Eventually they're going to help to transport the HN RNA which will become mRNA out of the nucleus into the cytosol. So they also play a little bit of a role in transport out of the nucleus and into the cytosol. Okay, so within this first step, what did we do?
We did 5'capping, we went over that part, and the 3'poly A tail that we did. Okay? Now we have this. So after we did all of this massive mess, we've come to this point.
Okay. On this part, what do we have? We're just going to write these, we're going to circle it here.
This is our... 5 prime cap with a 7-methylguanosine and on this end we already have kind of formed our poly A tail. The next thing that happens is what's called splicing and this can be sometimes a little annoying but it's not too bad I promise.
Let's say here this is the sequence of nucleotides within this RNA okay we're We're not at mRNA yet. We're still at this HNRNA. We're still kind of at this HNRNA at this point.
We haven't made mRNA yet. Within this HNRNA, there's particular nucleotides that will be red. translated and actually will code for particular amino acids.
There's other nucleotides within this ancient RNA that will not be read and they do not code for a particular amino acid. We give those very specific names. I'm going to highlight them in different colors.
So let's say I highlight this one here in pink. And then I will highlight this one here in this kind of maroon color. And then I'll pick here a blue.
And then we'll do another maroon color. And then we'll do one more color after this. Here's another maroon. And then we will do, just for the heck of it, black. Okay?
These portions here, the pink one, this is actually going to code for an amino acid. If it codes for an amino acid, we give a very specific name for that and we call it exons. So exons code. for an amino acid, okay?
Particularly amino acids which will make proteins. These other portions, and again, that's going to be, I'll mention which ones are exons and which ones are the next thing, which is called introns. Introns are basically nucleotide sequences that do not code for amino acids, which will help to make proteins.
Very important. I'm going to call this pink portion of the H and RNA. I'm going to call this an exon, but we have a bunch of them in this H and RNA.
So I'm going to call this exon 1. That's going to code for some amino acids. I'm going to have this portion here, which is going to be in the... I'm going to call this an intron, but you can have multiple introns.
So I'm gonna call this intron one Same thing here. This is going to be coding So if it codes it's what it's an exon, but we have multiple types. So we're gonna call this exon two two, then I'm going to test you again.
This one does not code for amino acids. So this is going to be a intron, but we have already intron ones. We're going to call this intron two.
And you guys already kind of get the pattern that I'm going with here. This one does code. So it's going to be a exon and we already have one, two.
So this will be three. Okay. We're going to do something called splicing where let's think about this.
If the introns don't code for any amino acids, amino acids, do we even need them? No. Let's get rid of them.
That's all that splicing is. It's getting rid of these introns, or also known as intervening sequences, and then stitching together the exons. Now, in order for that process to occur, we need very specific molecules, and we talked about it before. Let's see if you guys remember him. RNA polymerase 2 and 3, they made another very interesting small little RNA.
What was that all? RNA called? Small nuclear RNA, right? Original, right?
So small nuclear RNA is going to combine, we haven't used this color yet, so let's add this. These brown proteins, okay? So you're gonna have some proteins and some small nuclear RNA.
Together, these two things make up a very weird name called a SNRP. Okay, SNRPs, small nuclear ribonuclear proteins. So they're small nuclear ribonuclear proteins. And what they do is, these SNRPs are going to bind to this HNRNA. and they're gonna cleave out the introns in this actual RNA.
And then they're gonna stitch together the exons. So let's show that in a very basic way of how that happens. So these SNRPs, which are the SNRNA and your proteins are going to perform splicing. So what would that look like?
Let's take here our transcript here and bring it down here all the way down to this portion here. So here we're going to make our functional mRNA. So at this point in time we've actually made what at this point? We've made the mature mRNA. And if I were to show kind of what was the end result, what am I going to have here?
Let's say here I have a sequence that's in pink. That's exon 1. I got rid of entron 1, so what should be next? I should have exon 2. I got rid of Entron 2, so what should be left? We're going to expand it a little bit here. Get rid of that one.
Exon 3. So all I did was I took and got rid of each Exon, I mean each Entron. and stitch together only the exons. So now let's show kind of coming out of this process here.
What am I going to have kind of popping out off of this? The introns. And when the introns...
pop off, this can be intron 1, and then you can have another one, let's say intron 2. These are going to get popped off. We don't need these dang things anymore. So since we don't need them, we're just going to spit them off during that splicing process and only lead to the formation of axons.
Now within this mRNA, I have my 5'cap, I have my poly-A tail, I have only the nucleotide sequence which is going to code for amino acids. And then if you really want to go the extra mile, we said this is the 5'end, this is the 3'end. I'm not representing any kind of like dashes here. So this portion here and this portion here doesn't get translated or read at all by the ribosomes.
And so we call these regions, since they don't get translated, the 5'since it's near the 5'untranslated region. And this one doesn't get translated. So it's called the 3'. prime on translated region.
The only portion that gets translated is the exons. Now, I don't know why, but they love to ask this stuff in your exams, where you actually go through the specific mechanism of how the SNRPs truly do pull the introns out and splice together the exons. So let's say we take just exon 1. Let's write this one as exon 1 and this one is going to be exon 2. And then here in the middle we're going to make this intron 1. So let's kind of show you how these SNRPs, again the SNRPs, which is the SNRNA and the proteins, do this.
So if you really wanted to show it, let's just represent the SNRPs as kind of a black blob, if you will. They're going to kind of bind near this portion here. So here's my SNRP.
Within this portion here, right at the intron, at this portion, let's say here is the 3'end of this exon, 5'end of this exon. And let's say that here is going to be the 5'end of exon 2 and then the 3'end of exon 2. Okay? And then here's going to be the intron inside. Within this intron, you have a very specific nucleotide sequence that's near the 3'splice site near exon 1. 1 and the beginning of intron 1 and then you have a very specific nucleotide sequence near the 5 prime splice site at exon 2 and at the end of intron 1. What are that nucleotide sequence?
It's dumb but it helps me to remember it so I say I'm a G how about you? I'm a G. So you remember GU.
I'm a G how about you? That's the basic way that I remember the nucleotide sequence at the 3'splice site and then the one at the 5'splice site. Between exon 1, exon 2, and intron 1 in this example. There's another one. Right smack dab in the middle.
Let's make him a different color so we don't confuse it. Smack dab in the middle, there's a branch point, which is an adenine. Okay, there's an adenine right at this branch point.
And it has a very specific OH group kind of hanging from it. from it. Okay?
So this is called your branch point. What happens is, is the snurps will come in and they're going to cleave at that three prime splice site. Okay?
They're going to cleave this portion off. So what would that look like afterwards? So the snurps come in and they cleave at that three prime splice site.
And so what's going to be left over here is we're going to have Exon 1 somewhat separated here and coming off here, what's the 3'end contain? An OH group, right? And again, this is exon 1. Then the next thing you have here is intron 1. And it's...
It's going to have that kind of portion here kind of split off, if you will, right? It's kind of broken off here. And then again, over here, we're going to have still fused at this end, exon 2. So again, this is my 3'end, which has that OH.
This is the 5'end of that portion, of the exon. And then again, same thing over here. This is the 5'end of exon 2, 3'end of exon 2. And then again, what kind of nucleotides do we have in? here. We have that GU which was pretty much the marker which the snurp would cut at that three prime site.
Then on this five prime splice site I still have that AG and then here in the middle I have that branch point with the adenine with the OH group. Here's the next thing that happens. The OH group of that branch point will then bind or attack that GU site and pull it in to where it kind of fuses at this point. So it makes kind of like a little loop, if you will.
So let's show that. It attacks the GU and pulls it in. After that happens, you kind of form this weird little like loopy structure, if you will.
So what would that look like if we kind of drew after we had that attack? After that attack point, it's going to kind of look somewhat like this, if you will, where we have now that portion where, what would be here? What would be kind of the nucleotide sequence at that point right there?
G and U was attacked at that point by the OH at that branch point. Then the next thing happens. This is crazy. This 3'OH.
of exon 1 will then see that 5 prime splice site and it'll attack the 5 prime splice site at exon 2. When it attacks it, it then breaks away the nucleotide sequence, Ag, of this intron away from exon 2. So okay, now let's show what that would look like. So if the 3'OH attacks the 5'N, 3'OH of exon 1 attacks the 5'N of exon 2, now what do we have here? Exon 1 fused with exon 2. And we just fused the exons, and then what do we spit out after we break this off? The intron. Lariat, which I showed you like that before.
That is how this whole splicing process technically occurs. Super quick. Again, snurps bind. What do they do? Cut the three prime splice site between X on one.
intron 1. When it does that, the OH of the adenine at the branch point attacks the GU site, pulls it in, creates this loop. The 3'OH of exon 1 attacks the 5'N of exon 2. which snaps the intron out and stitches together exon 1 and exon 2. That is splicing. You're like, Zach, why the heck do I need to know all this crap?
There's a reason why. Whenever there's abnormalities within splicing, it can produce various amounts of diseases. Because think about it. If I don't cut out the introns properly, and I have introns mixed in with the exons, and introns don't code for amino acids, am I going to make a proper protein?
No, because I'm going to have areas that will code for amino acids and areas that don't code for amino acids. You know there's a very devastating condition called spinal muscular atrophy, where they are deficient in an SMN protein. You want to know why? Because the SNRPs aren't working properly. So there's a deficiency or there's a problem with the SNRPs not performing the proper splicing.
You know what else? There's another disease called beta thalassemia. Beta thalassemia, guess what?
You don't remove a particular intron and because you don't remove that intron you make a protein that's abnormal and it produces beta thalassemia. So there's reasons to know this stuff and again if someone has spinal muscular atrophy, do you know what that affects? The anterior gray horn neurons and then they develop lower motor neuron lesions, hypotonia, hyporeflexia, floppy baby syndrome, right? So it's a dangerous condition that can be traced back to something at the molecular level. All right, now that we talked about this, there's two more things and I promise we're done.
All right, so I want to talk about two more things and then we're done. The first thing I want to talk about, because it's very pretty much similar to what we talked about over here with splicing. I want to talk about something called alternative RNA splicing.
We understand the specific. reason for splicing. It's making sure that we only utilize exons to code for proteins, and there's no introns. Because if we have introns in there, it's going to freck up the whole protein production process. We'll get an abnormal protein.
With alternative RNA splicing, it gives variance of a protein. And I'll give you guys an example in just a second, but let me kind of talk about how this works. It's literally the same thing. We're not going to go too ham on this. Let's use the same colors here.
Here was Exon 1 and then here we had Intron 1 and we'll just skip this part here where that was Intron 2. And then blue here we had Exon 2 and then at the end here we had in black. Exon 3. Okay. So let's say that we take an example here of this kind of H in RNA, right?
So here's our H in RNA. And we want to make different mRNA. That'll give variants of proteins. So let's say here that we have, I'm just going to put exon 1, and I'm going to do all the same color here. Exon 2, exon 3, and then here in between we're going to have intron 1 and intron 2. Here's what I can do, which is really interesting and it's very cool when it comes to plasma cells and antibodies.
So let's say I use those SNRPs, right? So let's say here I put my SNRPs, right? My SNRPs, which are my small nuclear, ribonuclear proteins with the SNRNA in the proteins. They're going to splice, but they're going to do it in a very interesting way.
So let's say that the first one over here, we get the same thing that we did with that whole process of splicing, where we got rid of all the introns and we only have in exons. And let's say that we have exon 1. Let's say that we... we have exon two and then we have exon three, right? So we have all those exons here, exon one, exon two, and exon three. So we'll put these exon three, exon two, exon one.
So that's one, this is going to be mRNA, right? After we've kind of done that process and it'll give way to a particular protein. And we'll call this protein. A, if you will.
Okay? Then we're going to go through the same thing. The SNRPs are going to cleave out the N-trons and only leave in the exons. But let's say for this example we pop out, so this one we popped out N-trons, but let's say with this one we pop out both the N-trons and let's say that we pop out exon 2. Let's say that we don't want exon 2 in this one. So then what am I going to be left with?
I'm going to be left with only exon 1 and exon 3. And by doing that, that's going to give me an mRNA that'll code for another protein. Let's call this protein B. And then last but not least, you guys can already probably see where I'm going with this.
Let's say that this last one, example 3, again, we cut out the introns. We always got to cut out those introns. But in this case, we cut out exon 3. I don't want to cut out the introns.
want that one in the diagram. I don't want this one in that mRNA. So what am I left with?
I'll be left with exon 1 and I'll be left with exon 2. And what will this code for? This will code for, this will give an mRNA. That'll then do what? Code for another protein, and let's call this protein C.
From one HN RNA, we made three different mRNAs and made three proteins. from the same h and rna or from the same kind of a gene if you will that means it's going to be the same protein if it's coming from the same gene but it's a variant of that protein you know what this is examples of think about it guys think about plasma cells which make antibodies when they make antibodies you can have antibodies that can be secreted or you can have antibodies that are different and they're expressed on the cell membrane that could be one example so antibodies differences and antibodies would be an example of how that works from alternative RNA splicing because I'm making one protein that will bind to the membrane and one protein that can be secreted think about neurons Let's say here's one neuron, and this neuron has a dopamine receptor, dopamine 1 receptor. But then you have another neuron, and this has a dopamine 2 receptor. It's the same gene that's making these proteins but just a variant of it.
And then the last thing is take an example of a muscle within the heart. called tropomyosin and the muscle, and then within the skeletal muscles, tropomyosin. They're different. They're small changes or variants within the protein that are coming from the same gene.
So one of the things that they'll love to ask on your exam questions is alternative RNA splicing gives you, takes one gene, one HNRNA, gives you multiple mRNAs in variants of the same protein. If you give examples, something like immunoglobulins, dopamine receptors of the brain, or... Tropomyosin variant within cardiac and skeletal muscle.
Alright, NadegeNerds, I promise I'm so sorry for this being so long, but there's one last thing that I want us to talk about. The last thing that I want us to discuss is called RNA editing. This is also mentioned a lot in your exams, and the reason why is...
It's really interesting kind of how this happens. There's two different types of RNA editing. I only want to mention really one of them because it's the most relevant to your USMLEs and kind of a clinical setting. So let's say here we have our mRNA, right? So this isn't an hRNA.
We've already at this point in time for RNA editing, we've already formed our functional mRNA. So at this point in time, this structure here is a mRNA, okay? This mRNA can have a particular nucleotide sequence that a special enzyme can read and sometimes switch nucleotides with. What is that nucleotide sequence which can be seen in this mRNA which we really want to know?
It's CAA. We're going to be talking about apoproteins that's why I'm mentioning CAA. So this is our signal which is really really important within this mRNA. which is going to be making apoproteins, a particular protein called, let's say that this mRNA is going to code for a particular protein called APOB100.
If you guys watch our lipoprotein metabolism video, this will sound familiar, right? But APOB100, this is going to be the mRNA that will code for that protein. And here's a particular nucleotide sequence that we're going to modify. In the hepatocytes, this nucleotide sequence is not altered in any way. It's kept the same.
So it's not going to be changed. It's still going to be CAA. And whenever this mRNA is translated by ribosomes, it makes a particular protein that we already talked about called APOB100.
But in enterocytes, okay, your GI cells, what are these cells here called? These are called your enterocytes. They have a very special enzyme where they can modify the same gene that makes ApoB100 but make a different protein. How the heck?
How do they do that? Let me explain. There's this cute little blue enzyme in the enterocytes called cytidine. deaminase. And what this cytidine deaminase does is, is it deaminates the cytidine right here, or the cytosine nitrogenous base, and switches it with uracil.
So now let's switch it here where we're going to have this as switching C and putting UAA. If you guys know anything about your codons, There's a little trick to remember your stop codons. Do you guys remember the little way to remember them?
You remember it by You go away. You are away. You are gone These are the easy ways to remember your stop codons. Does any of of these look like a stop codon?
Yes. UAA, that's a stop codon. So what's going to happen is when you have the ribosomes, which will be reading this, let's say here I kind of put like a little ribosome, it's going to be reading this and making a particular protein. As it gets to this point where it's going to translate it, that's a stop codon. Will it then read the rest of the RNA and translate that into a long protein?
No. So at this point, translation will stop. You won't read all the rest of the mRNA and make the full protein.
Instead, you'll make a smaller protein. And this small protein is called APOB48. This is something that they love to ask on your exams because you're taking the same mRNA, just modifying it a little bit to produce a different protein. That is a completely different sized protein.
So that's really cool. Definitely wanted you guys to know that. And that finishes our lecture on DNA transcription.
All right, Ninja Nerds. So in this video, we talk a ton about DNA transcription. I hope it made sense and I hope that you guys did enjoy it. As always, Ninja Nerds, until next time.