Eukaryotic Gene Expression – Complexity of RNA Structure

by Kevin Ahern, PhD

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    00:01 In this presentation we'll begin to get an idea of the complexity of RNA, and also the complexity and the ways that RNAs are made.

    00:08 I will be covering the topics here of Eukaryotic gene expression, and RNAs and RNA polymerase as we shall see.

    00:16 Now, in eukaryotes, the scenario for making RNA is very complicated.

    00:21 The proteins that cover the DNA making up the protein DNA complex called chromatin, have a major role in basically hindering gene expression.

    00:32 So in order for genes to be able to be expressed in eukaryotes, they have to not only be able have the polymerase find the promoter, but they also have to be able to open up a region so that promoter can be found.

    00:44 And that's what I want to talk a little bit about here.

    00:47 So the complexity is enormous.

    00:49 And moreover, the sequences are larger, and the genes are very widely spaced apart.

    00:56 So chromatin, to define it, is a complex of DNA, and histone proteins that make up what we call the chromosomes.

    01:04 Now, the histone proteins are positively charged small proteins that the DNA wraps with as we shall see.

    01:11 In order to help the DNA polymerase to find the right sequence, proteins called transcription factors help facilitate this process.

    01:21 And they work in a variety of ways.

    01:23 And one of the ways I'm going to discuss here is through the use of enhancer sequences that they bind to to help set up the transcriptional machinery.

    01:33 Now, chromatin is the first thing I need to discuss because chromatin is really a complex that has to be dealt with in order for RNA to be made.

    01:41 As I said, it's a complex of DNA, protein, and in some cases, actually RNA comprising the eukaryotic chromosomes.

    01:48 For RNA polymerase to perform this transcription, this chromatin must be changed so that access can be gained to the DNA.

    01:58 Now, to give you an idea of this complexity, I've presented these images below.

    02:03 And they start on the left with a very far out view of chromosomes as might be seen in a microscope.

    02:10 And then sequentially, I zoom in closer, and closer, and closer ultimately getting to the individual DNAs.

    02:16 So if we start on the far left, we can see what are called metaphase chromosomes where we have just the basic coils that are there.

    02:24 And these would be visible in a visible light microscope.

    02:27 Zooming in a little bit further, we can see these coils have some various things that help hold them together.

    02:33 And moving even further, we get to an interface chromosome where we can see a part of the chromosome that might be involved in making RNA.

    02:42 As we look closer, we see the loops of the individual chromatin fibers becoming apparent.

    02:50 The fibers have a structure called the 30 nanometer fiber that you can see here, from a distance and zooming in even closer, you can now see that they have so very tight organization that's there.

    03:02 And actively transcribing set of genes will have a DNA region that's described as beads on a string, sort of like what you see here.

    03:10 And examining those beads on a string closer, we discovered that they're made up of DNA coiled around these individual histone proteins that I described.

    03:21 And of course, if we peel away the histone proteins, we're left with bare DNA.

    03:28 Now, a nucleosome is a term that we need to be familiar with.

    03:32 That looped structure that I described on the last figure is called a nucleosome.

    03:36 So it's the simplest unit of chromatin structure.

    03:39 And you can see it here.

    03:41 Within this loop, you see various colored proteins.

    03:45 And there are in fact eight proteins found within that loop.

    03:49 There are two copies each of four histone proteins called H2a, H2b, H3, and H4.

    03:58 The DNA is wrapped around this core, as you see here.

    04:01 And then there's an additional protein called H1 that's on the outside.

    04:05 And you can actually see this wrapping occurring here.

    04:09 Now you'll notice that the proteins are positively charged, and that helps them to interact with the DNA because the DNA backbone is negatively charged.

    04:18 We can also think of the histone H1.

    04:20 It is kind of sealing and holding this structure together.

    04:23 It helps to stabilize the nucleosome.

    04:26 Now an important point that I mentioned is that the histone proteins are positively charged.

    04:32 This means that they're rich in the basic amino acids, typically arginine and lysine.

    04:37 And in order to change the structure of the chromatin, we have to change those positively charged, because that strong attraction between the positive and the negative is what holds them together very tightly.

    04:49 And tight access of these histone proteins to the DNA actually inhibits the process of transcription.

    04:57 So we can imagine now that this type core has to be sort of loosened up, or given access to the transcriptional machinery, so that transcription can happen.

    05:07 Chemical modifications actually happen that allow these changes to occur in transcription.

    05:13 And the chemical modifications that occur change the positive charges.

    05:18 A group of enzymes called the Histone acetyl transferases or HATs used acetyl-CoA to cover up some of the positive charges of the lysine residues that are in histones.

    05:29 This action has the effect of neutralizing their negative charge.

    05:32 Because by putting these acetyl groups on there, the charge of the side chain of lysine changes from being positive to being charged zero.

    05:40 This zero charge as you might imagine, allows for a sort of loosening of the interaction that's between the histone proteins and the DNA.

    05:48 This sort of loosening interaction is called remodeling or restructuring of the chromatin.

    05:52 And it must happen in order for transcription to occur.

    05:56 We can see the duplex below round around the histones loosening up with the chemical modification, which leads to the scenario on the right.

    06:03 Instead of having many coils together, we have individual coils like the beads on a string that we saw earlier.

    06:10 In addition to having their charges changed and allowing this opening up as you see here, the acetylated lysine can be a specific target for proteins that affect transcription.

    06:19 One reason this happens is because the DNA is open.

    06:22 Another reason is that if that acetyl group is a target for a protein, it's also helping to focus the transcriptional machinery to come to the place where transcription should occur.

    06:32 Acetyl-CoA is also used to cover up the positively charged residues of lysine.

    06:38 Now, histone acetylation favors what we call euchromatin.

    06:42 So euchromatin is a portion of the overall chromatin that is the part of the chromosome that is transcriptionally active.

    06:50 So acetylation favors transcription.

    06:53 We can see this difference here in this schematic at the lower right of the screen.

    06:58 We see what's called heterochromatin, which was the structure that we had before the acetylation.

    07:03 And we see the euchromatin, which is what we have after it.

    07:07 Euchromatin is described as active and heterochromatin is described as silent.

    07:12 If acetylation favors the formation of euchromatin, then removal of the acetyl group is essential for the formation of heterochromatin.

    07:22 So there are enzymes called histone de-acetylases that reverse the effects.

    07:26 And they do that by removing the acetyl groups from the side chains of lysine.

    07:31 Now, the lysine becomes positively charged and this ordered structure called heterochromatin forms.

    07:38 That heterochromatin will not be transcriptionally active.

    07:41 So in eukaryotes, the ability to make RNA is a function of whether the chromatin is in the euchromatin state, or the heterochromatin state.

    07:55 Now, there are numerous modifications that are made to histone proteins.

    07:59 I don't want to leave the impression that acetylation is the only one that happens.

    08:02 In fact, in a figure I'll show in a minute, there's a lot of different things that can happen to individual histone proteins.

    08:10 These include acetylation and deacetylation, methylation and demethylation, that is putting methyl groups on.

    08:16 Phosphorylation and dephosphorylation, and ubiquitination.

    08:21 So each of these individual modifications can affect the individual histone proteins.

    08:27 Now, this talk, I won't be able to go through all of the different scenarios that are there.

    08:31 But you can imagine that these modifications are there to help facilitate either the heterochromatin state or the euchromatin state.

    08:39 The chemical modifications to bases in DNA can also affect this process.

    08:44 And we'll see how that comes up in a later slide.

    08:48 So this slide in this very complex slide, We can relax.

    08:51 I'm not going to take you through it.

    08:53 But this very complex slide shows for each of the individual proteins in the histone core, that is the core of eight proteins in the center of the nucleosome.

    09:03 You can see the individual modifications that can happen to each of those proteins.

    09:09 Now, eukaryotes are very complex. They need a lot of controls.

    09:14 They're different from E. coli.

    09:16 E. coli has some pretty simple needs.

    09:18 Do I have energy? Do I need energy? Do I have lactose? Do I need lactose? In eukaryotic cells there are fine levels of control that have to be maintained.

    09:29 Am I a skin cell? Am I a muscle cell? Am I differentiating? Will I become something else? What are my needs right now? So very, very widely ranging needs.

    09:41 And these widely ranging needs are very important to be controlled properly.

    09:46 So like we saw in another presentation controlling the lactose operon of E. coli under very simple circumstance, the number of circumstances that a eukaryotic cell finds itself.

    09:57 And they are amazingly complex.

    09:58 And that's why we see the complexity of these controls on the screen.

    About the Lecture

    The lecture Eukaryotic Gene Expression – Complexity of RNA Structure by Kevin Ahern, PhD is from the course RNA and the Genetic Code.

    Included Quiz Questions

    1. Histone H1 is found in the center of the octet core
    2. It contains two copies each of histones H2a, H2b, H3, and H4
    3. DNA is wrapped around the histones
    4. The negatively charged DNA is attracted to the positively charged histones
    1. It causes them to be less positively charged
    2. It occurs on glycine side chains
    3. It causes transcription to be inactivated
    4. It creates heterochromatin
    1. Binding and set up of the transcriptional machinery
    2. Deacetylation of the H1 protein
    3. Ubiquitination of H2A and H2B proteins
    4. Binding of helicase and topoisomerase enzymes along with DNA polymerase at the centromere of the chromosome
    5. Binding of the nucleases to the damaged DNA sequence
    1. Histone deacetylase
    2. Topoisomerase
    3. Nuclease
    4. Phosphorylase
    5. Phosphofructokinase

    Author of lecture Eukaryotic Gene Expression – Complexity of RNA Structure

     Kevin Ahern, PhD

    Kevin Ahern, PhD

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