DNA Transcription - Online Tutor, Practice Problems & Exam Prep

Transcription is the process of converting DNA into RNA, primarily mediated by RNA polymerase. In prokaryotes, transcription can produce polycistronic mRNA, while eukaryotes generate monocistronic mRNA. Key elements include the transcription start site, promoter sequences, and transcription factors like TFIID and TFIIH, which facilitate RNA polymerase binding and activation. During elongation, RNA polymerase synthesizes RNA in a 5' to 3' direction, forming phosphodiester bonds. Termination occurs when RNA polymerase reaches a terminator sequence, leading to the release of the RNA transcript and the enzyme's dephosphorylation.

Transcription Overview

Hi, in this video, we're going to be talking about DNA transcription. So in this video, we're really going to be just doing a brief overview of some concepts that we need to know about transcription before we get into more nitty-gritty details. So, transcription is the process that changes DNA to RNA. And so, how is that process catalyzed? Well, that's through enzymes known as RNA polymerase, and RNA polymerase is the class of enzymes that transcribe DNA. So, I made this little table. This has, you know, RNA polymerase, what organism it's in, and then also what it transcribed. So, the first one, which I've already highlighted, is just called RNA polymerase. That's in prokaryote, and it does all prokaryotic RNA. Now, eukaryotes are a little more complex. And so, they actually have three which are very easily labeled RNA polymerase 1, 2, and 3. And they all work in eukaryotes. But because there are three different classifications, that means they do different types of RNA. RNA polymerase 1 focuses on rRNA. RNA polymerase 2 focuses on mRNA. And RNA polymerase 3 focuses on tRNA. Now, we're going to be talking a lot about transcription. And most of our focus on transcription will be focusing on mRNA. And that's the RNA that forms proteins. So, RNA polymerase 2 is going to be a big player. You need to know about the other ones, really, that they exist and they do other types of RNAs, but we're not going to be focusing on them as much. So, how does transcription work? Well, transcription uses one strand of DNA as a template to produce a single-stranded RNA. And so, this DNA template can encode for one gene or it can encode for multiple. So, for prokaryotes, they're called polycistronic, meaning that a single DNA that's transcribed into a single RNA can encode for multiple genes. Whereas eukaryotes are a term called monocistronic, which means that the single RNA encodes for only one gene. So, just an example of this down here, let me move out of the way. You have your monocistronic here, which encodes for one gene, and your polycistronic here, which encodes for multiple genes. And this is on a single RNA transcript. Now, one thing to know is when we're talking about RNA transcription, we're talking about RNA polymerase, and of course, it's not perfect. Nothing in cell biology is just absolutely perfect. There are errors, and these errors for RNA polymerase are about one mistake every \(10^4\) nucleotides, which is not nearly as accurate as the DNA polymerase, which is responsible for replicating DNA. It will be replicating, and that's actually \(10^7\). So, it's actually much worse than the DNA polymerase that replicates DNA. But that's okay because it gets the job done. And so yeah, that's transcription. So now let's turn the page.

Transcription Initiation

Okay, continuing on this page, we are going to be talking a lot about transcription initiation. Now, you're going to have to bear with me; there's a lot of content and it's especially a lot of vocabulary, but I'm going to walk through all the vocabulary slowly, but just sort of get ready. We're going to learn a lot of terms in this video. So the first term we're going to learn is actually called the transcription start site. And the transcription start site is important because it tells RNA polymerase where to start, which makes sense—it's in its name.

What is part of a transcription start site? Well, one of the things is called a promoter sequence and this is kind of a vague term and essentially it's just a specific set of nucleotides that RNA polymerase can bind to in order to start. So these are the sequences that say, you know, "bind here." This is where you need to start transcribing. So it's kind of interesting because you would think in order to start transcribing it can only lie upstream, but actually it can also lie downstream. And so, I've given a couple examples of promoters here; we're going to actually talk about these in just a few minutes, but just sort of as I'm introducing these names—upstream or downstream.

Now, we talk mainly about upstream promoters because those are the ones that make the most sense, and there are also the most of those. There are two types of upstream promoters and these are proximal control elements, and so these are elements of sequences that are located really close to the gene start site. So, 100 or 200 nucleotides away. But then there's a second kind; those are called enhancers. And these are located far away from the gene start and, again, these can be upstream or downstream. And so, how do we determine what these sequences are? Are these sequences completely identical in every organism or even between humans? No, they're not completely identical. So, we don't call them conserved sequences because conserved sequences are practically identical, but we can call them consensus sequences.

What is a consensus sequence? Well, that is going to be a common version. So, they're not completely identical, but we continually see these repeating patterns of sequences over and over again in these regions. And I'll show you an example of that in just a minute. Now, we have these sequences and that's great. But how does the polymerase actually recognize these sequences? Well, the polymerase can find the promoter, the promoter sequences because those sequences give the helical backbone of the DNA unique features. So these can be unique properties or charges or polarity or structures. But these sequences provide these very unique backbone structures and the RNA polymerase says, okay well that's where I've got to bind because that's the charge I'm looking for, or that's the polarity I'm looking for. And so, we call this promoter has polarity which is a little misleading because we're not talking about its interaction with water but instead we're talking about the fact that the promoter can only binds the wrench side. So the DNA is double-stranded but the gene is really only on one side of the DNA. The other side encodes a completely different thing. And so when the RNA polymerase binds it has to bind to the correct strand. So we're saying that the promoter has polarity; that promoter structure or sequence or charge or whatever is recruiting the RNA polymerase is only that way on one side. So it positions the RNA on only one strand in one direction.

If we're to look at what this would look like. So, we're going to say this black thing is that chromosome, sort of a section of a chromosome. What you see here is you have your gene, whatever you're trying to transcribe, and then you have proximal control elements here. These are close. So we call these the promoter with proximal elements. And then way over here you have your enhancers, and these are really far away. And so you can see that the factors are in a polymerase, for instance, or other factors that we'll talk about in a second that are important for starting transcription. Get recruited to these sites and allow for transcription to occur. Let me back out of the way transcription. So, like I said, these elements don't have to always be upstream and they don't have to be nearby. They can be far away and they can be upstream or downstream.

Now let's come back; we'll talk about some factors that are important for recognizing this promoter and binding to it and sort of recruiting this RNA polymerase and allowing transcription to occur. Like I said, a lot of vocabularies but just kind of bear with me and we'll go through it slowly. So, the first thing is that prokaryotes are of course different than eukaryotes. So, the important word that you need to know about prokaryotic transcription is called sigma factor and this is actually a region on the RNA polymerase itself and that is the region that binds promoter RNA. There are multiple types of sigma factors. Each of them binds to different promoters. And so that's kind of how prokaryotes control which genes are going to be transcribed. It's simple, it makes sense. You know you want to bind a different promoter, use a different sigma factor. But for eukaryotes of course, it can't be that simple; never is. So for eukaryotes, we use things called transcription factors and what these do is these are proteins that bind the promoter and recruit RNA polymerase and so we need these factors to not only just sort of control gene expression but also to recruit RNA polymerase to the correct gene that needs to be transcribed because we don't need to be transcribing all genes at all times. So these transcription factors really give us control over gene transcription. So, I've mentioned a couple of these before but we're going to go through exactly what these sequences are and what they mean and what they do.

So, the first two main sequences that are really important to control, you know, promoter binding and transcription initiation. One of them is called the TATA box or TATA box, however, you want to say it. But it essentially it's called that because that's what it is. It's a sequence of TATA. And this is bound by a transcription factor known as TFIID. Now there's a second one and this is the INR, initiation sequence. And you could use this, or the promoter RNA polymerase can use the sequence with or without the TATA box. So the TATA box doesn't always have to be present. But the INR sequence is always present. So you're always going to have this and this most of the time but not always. And so, what happens? So we have these sequences; they are, you know, in the right position and they begin recruiting transcription factors like I said this TFIID. So, once the TFIID is there, it as a protein starts recruiting this other complex known as the transcription initiation complex. And this is a collection of proteins that need to be at the promoter to promote transcription and what the, what's the collection of proteins? Well, I wish it were simple but it's not because it differs for every gene and that kind of makes sense because every gene needs to be regulated and transcribed differently. So of course, you need to regulate that transcription with a different collection of proteins. But just for simplicity's sake, we call all of these initiation complexes transcription initiation complexes and they contain a lot of protein sometimes. I mean over 100; it's kind of a complex process now. We've talked about TFIID, but there's another one that gets recruited in this initiation complex called TFIID and this is really important because it contains a protein kinase. Which if you remember what a protein kinase is great, if you don't, I wrote it down, it adds a phosphate. And that's really important because RNA polymerase has to have this phosphate before it can work. So if TFIID is not there, it's not going to get phosphorylated. And it's not ever going to activate. So it needs that. So we have all these. So I mean it's kind of hard to imagine. There's all these different terms. Like I said, it was going to be big on vocabulary. But you can kind of understand how this works. There are these sequences on these chromosomes. They recruit different transcription factors, especially TFIID. Once TFIID is there, that recruits a bunch of other factors. And one of them, TFIID, is responsible for adding a phosphate to RNA polymerase which says go-ahead, get started ready to transcribe. And so, once transcription begins, it makes sense the transcription factors are no longer needed. And so they actually, whoops let me not highlight that leave the promoter. So this all is sort of in a stepwise process. We're going to go over a lot of steps in cell biology. So we're just getting started here, now there's another vocabulary word. Of course. Not that easy and not done yet. And this is called a mediator or mediators. And this is just another group of proteins. Or another set of proteins that are found in this transcription start area and they communicate between RNA polymerase and different transcription factors and transcription. You know, mediators to really just sort of get everything communicating together. So transcription can be activated. So a lot of processes here. Now this is all for RNA polymerase II do you remember was the one I said that we were going to be talking a lot about but there are RNA polymerase I and RNA polymerase III as well. And of course, this process differs from them. And they have different promoters and different transcription factors that really drive this process. But for now, we're going to focus on RNA polymerase II because that's really important and that's the one that drives everything we're going to care about. At least for the most part at least for this part of cell biology. So they back up and I'll show you this example. So this is a promoter element and consensus sequence which I said I was going to show you on a DNA region. So let me walk through this for a second. So here you have numbers. So what do these numbers mean? These are positions of nucleotides to the gene. So you'll notice here that the gene isn't anywhere on here but it's just all these control regions. So the number tells you where the gene is going to lie. So it's going to lie at zero. That's going to be the start site. So if I was adding the gene here, the gene would start here and go that way. Now you'll notice that one of the important transcription initiation sites or promoter sites is the initiator. And it actually overlaps with the gene. And this is true for most initiation sites. And so you'll also see here that this gene particularly has the TATA box. It also has this other element that we didn't talk about. But just giving you an example, we didn't talk about every promoter that there is, just a couple of the important ones, but just know there's all these other ones. And just for good measure here's a downstream promoter element which is actually downstream of the gene. Now the consensus sequences here and you'll see that this isn't a conserved sequence because it's not exactly the same in all organisms. But what you can see is that there's this consensus, you know, it's either going to be a GRC, it's either going to be a GRA, it's either going to be a GA or C in this position, so it's not identical. There is this flexibility that exists in consensus sequences that isn't there in conserved sequences. So promoters generally have consensus sequences. So, with that let's now move on.

Transcription Elongation

Okay, so now we've talked about transcription initiation, but let's talk about transcription elongation. After RNA polymerase gets started, it has to transcribe the whole gene. It has to work continually to stay on and elongate the RNA transcript so that the entire gene is transcribed. How does it do this? There are proteins that travel with RNA polymerase, bind to the RNA polymerase, and continually open up the DNA strand so that RNA polymerase can continually transcribe the gene it is working on. One of these proteins, which we've already discussed, is TFIIH. It travels continually and is responsible for opening around 12 to 14 base pairs at a time, allowing RNA polymerase to continue down the strand.

Now, how does RNA polymerase actually transcribe DNA to RNA? It is responsible for catalyzing phosphate diester bonds between ribonucleotides during transcription. Recall our earlier discussions about phosphate ester bonds and the bonds that form between RNA and DNA and different nucleotides. RNA polymerase is the enzyme that catalyzes these bonds. These reactions do not require an input of energy because they are energetically favorable, and they end up releasing two phosphates. Although you do not need to know the specifics of the two phosphates, it is important to understand that these are energetically favorable reactions that do not require additional energy but are still quite overwhelming. Considering the number of nucleotides in every single gene, these nucleotides are added one at a time, but very quickly, at a rate of approximately 1000 nucleotides per minute. This is a tremendous amount of nucleotides to process in a single minute. As mentioned, these factors move along with the RNA polymerase, help keep it attached, help it catalyze reactions, and help open the DNA so it has access to it. All these factors are known as elongation factors because they assist RNA polymerase in elongating the transcript.

Just as a quick reminder of what phosphate diester bonds are: here, we have different nucleotides, and you can see the phosphate diester bonds forming between them. This is the function of RNA polymerase. Another key function of RNA polymerase is to catalyze RNA transcription in a 5 prime to 3 prime direction. Remember, the DNA has different orientations of the DNA backbone on each strand. This means that to transcribe in a 5 prime to 3 prime direction, it must bind on the 3 prime side, which allows it to transcribe in the correct direction. As a result, you obtain an RNA transcript that also runs 5 prime to 3 prime. However, to achieve this, it must bind to the 3 prime end to transcribe appropriately.

That was an overview of transcription elongation. Let's move on.

Transcription Termination

Okay, so now we're going to talk about transcription termination. So far, you know RNA polymerase has been chugging along, it's been transcribing and elongating the transcript but it's done. And so, how does it know it's done? Well, it knows it's done because it reaches a terminator which is generally just a stop site, a stop sequence for transcription. So when it reaches this terminator, what happens is the phosphates on the tail are removed. So, if you remember, in order for RNA polymerase to initiate, it had to have a phosphate on it. And so in order to stop, that phosphate has to be removed. And so that gets removed by protein phosphatases which are enzymes that remove phosphates.

Now, the newly dephosphorylated, or the protein or the enzyme without phosphates, is free to go somewhere else, go to a different gene, become phosphorylated again and start again. It doesn't need any kind of extra things. As soon as it's done, it can start all over. So, if we're to look at this, let me move out of the way. You've seen a similar picture to this before where you have RNA polymerase but now I've added a phosphate. You can see here, so it's transcribing this way and it eventually reaches some kind of termination sequence or terminator and that sequence releases RNA polymerase from its phosphate and then that is free to go transcribe somewhere else wherever it wants to. So that's transcription. So now let's move on.

Transcription initiation requires many factors, which of the following is not one of them?

• A. RNA polymerase
• B. Mediator
• C. Inr initiation sequence
• D. Elongation factors

Is polycistronic mRNA found in prokaryotic transcription or eukaryotic transcription?

• A. Prokaryotic
• B. Eukaryotic

Transcription occurs in which of the following directions?

• A. 5' to 3'
• B. 3' to 5'
• C. 3' to 3'
• D. 5' to 5'

When RNA polymerase reaches a terminator, what happens?

• A. It immediately falls off the DNA sequence
• B. It is marked by a protein that removes it from the DNA
• C. Phosphates are removed from the polymerase tail, which cause it to remove from the DNA
• D. It remains on the DNA strand, but does not transcribe

Both strands of the DNA are transcribed to create a protein.

• A. True
• B. False

Here’s what students ask on this topic:

What is the role of RNA polymerase in DNA transcription?

RNA polymerase is the enzyme responsible for synthesizing RNA from a DNA template during transcription. It binds to specific promoter sequences on the DNA, unwinds the DNA helix, and catalyzes the formation of phosphodiester bonds between ribonucleotides. This process occurs in a 5' to 3' direction, producing a single-stranded RNA molecule. In prokaryotes, a single RNA polymerase transcribes all types of RNA, while in eukaryotes, there are three types: RNA polymerase I (rRNA), RNA polymerase II (mRNA), and RNA polymerase III (tRNA). RNA polymerase II is particularly important for synthesizing mRNA, which is later translated into proteins.

What are the differences between prokaryotic and eukaryotic transcription?

Prokaryotic transcription involves a single RNA polymerase that transcribes all types of RNA and often produces polycistronic mRNA, which can encode multiple genes. In contrast, eukaryotic transcription is more complex, involving three different RNA polymerases: RNA polymerase I (rRNA), RNA polymerase II (mRNA), and RNA polymerase III (tRNA). Eukaryotic mRNA is typically monocistronic, encoding only one gene. Additionally, eukaryotic transcription requires various transcription factors and occurs in the nucleus, whereas prokaryotic transcription occurs in the cytoplasm and involves simpler regulatory mechanisms like sigma factors.

What is the function of transcription factors in eukaryotic transcription?

Transcription factors are proteins that bind to specific DNA sequences, such as promoters and enhancers, to regulate the initiation of transcription in eukaryotes. They help recruit RNA polymerase to the correct gene, ensuring that only the necessary genes are transcribed at the right time. Key transcription factors include TFIID, which binds to the TATA box, and TFIIH, which has kinase activity that phosphorylates RNA polymerase II, activating it for transcription. These factors form part of the transcription initiation complex, which is essential for the precise regulation of gene expression.

How does RNA polymerase recognize promoter sequences?

RNA polymerase recognizes promoter sequences through specific DNA motifs that provide unique structural and chemical features to the DNA helix. These features include unique charges, polarities, and structures that are recognized by the RNA polymerase or associated transcription factors. In eukaryotes, transcription factors like TFIID bind to the TATA box, a common promoter element, and help recruit RNA polymerase II to the transcription start site. The promoter's polarity ensures that RNA polymerase binds to the correct DNA strand and initiates transcription in the proper direction.

What is the significance of the TATA box in transcription initiation?

The TATA box is a conserved DNA sequence found in the promoter region of many eukaryotic genes. It is typically located about 25-30 base pairs upstream of the transcription start site. The TATA box is recognized and bound by the transcription factor TFIID, which helps recruit other components of the transcription initiation complex, including RNA polymerase II. This binding is crucial for the accurate initiation of transcription, as it positions RNA polymerase II correctly on the DNA template, ensuring that transcription starts at the right location.