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Lecture 19: Translation & post-translational processes - Biology

Lecture 19: Translation & post-translational processes - Biology


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Lecture 19: Translation & post-translational processes

Lecture 13: Gene Regulation

Download the video from iTunes U or the Internet Archive.

Topics covered: Gene Regulation

Instructors: Prof. Eric Lander

Lecture 10: Molecular Biolo.

Lecture 11: Molecular Biolo.

Lecture 12: Molecular Biolo.

Lecture 13: Gene Regulation

Lecture 14: Protein Localiz.

Lecture 15: Recombinant DNA 1

Lecture 16: Recombinant DNA 2

Lecture 17: Recombinant DNA 3

Lecture 18: Recombinant DNA 4

Lecture 19: Cell Cycle/Sign.

Lecture 26: Nervous System 1

Lecture 27: Nervous System 2

Lecture 28: Nervous System 3

Lecture 29: Stem Cells/Clon.

Lecture 30: Stem Cells/Clon.

Lecture 31: Molecular Medic.

Lecture 32: Molecular Evolu.

Lecture 33: Molecular Medic.

Lecture 34: Human Polymorph.

Lecture 35: Human Polymorph.

Good morning. Good morning.

So, what I would like to do today is pick up on our basic theme of molecular biology. We've talked about DNA replication.

The transcription of DNA into RNA, and the translation of RNA into protein. We discussed last time some of the variations between different types of organisms: viruses, prokaryotes, eukaryotes, with respect to the details of how they do that in general that bacteria have circular DNA chromosomes typically that eukaryotes have linear chromosomes, etc. What I'd like to talk about today is variation, but variation not between organisms but within an organism from time to time and place to place, namely, how it is that some genes or gene activities are turned on, on some occasions, and turned off on other occasions. This is, obviously, a very important problem to an organism, particularly to somebody like you who's a multi-cellular organism, and has the same DNA instruction set in all of your cells.

It's obviously quite important to make sure that the same basic code is doing different things in different cells.

It's important, also, to a bacterium to make sure that it's doing different things at different times, depending on its environment. So, I'm going to talk about a very particular system today as an illustration of how genes are regulated, but before we do that, let's Ask, where are the different places in this picture?

DNA goes to DNA goes to RNA goes to protein, in which you might, in principle, regulate the activity of a gene. Could you regulate the activity of a gene by actually changing the DNA encoded in the genome? So, why not? Because what? It becomes a different gene. Yeah, that's just a definition.

Why couldn't the cell just decide that I want this gene now to change in some way? Oh, I don't know, I'll alter the DNA sequence in some way. And, that'll make the gene work.

Could that happen? Is that allowed? Yeah, it turns out to happen.

It's not the most common thing, and it's not the thing they'll talk about in the textbooks a lot but you can actually do regulation.

So, the levels of regulation are many, and one is actually at the level of DNA rearrangement. As we'll come to later in the course, for example, your immune system creates new, functional genes by rearranging locally some pieces of DNA, some bacteria, particularly infectious organisms control whether genes are turned on or off by actually going in there, and flipping around a piece of DNA in their chromosome.

And, that's how they turn the gene on or off is they actually go in and change the genome. There's some protein that actually flips the orientation of a segment of DNA. Now, these are a little funky, and we're not going to talk a lot about them, but you should know, almost anything that can happen does happen and gets exploited in different ways by organisms.

So, DNA rearrangement certainly happens. It's rare, but it's always cool when it happens.

So, it's fun to look at. And, something like the immune system can't be dismissed as simply an oddity. That's an incredibly important thing. The most common form is at the level of transcriptional regulation, where whether or not a transcript gets made is how it's processed can be different. First off, the initiation of transcription that RNA polymerase should happen to sit down at this gene on this occasion and start transcribing it is a potentially regulatable (sic) step that maybe you're only going to turn on the gene for beta-globin and alpha-globin that together make the two components of hemoglobin, and you're only going to turn them on in red blood cells, or red blood cell precursors, and that could be done at the level of whether or not you make the message in the first place. That's one place it can be done.

Another place is the splicing choices that you make.

With respect to your message, you get this thing with a number of different potential exons, and you can regulate how this gene is used by deciding to splice it this way, and skip over that exon perhaps, or not skip over that exon. That alternative spicing is a powerful way to regulate. And then finally, you can also regulate at the level of mRNA stability.

Stability means the persistence of the message, the degradation of the message. It could be that in certain cells, the message is protected so that it hangs around longer.

And, in other cells, perhaps, it's unprotected and it's degraded very rapidly. If it's degraded very rapidly, it doesn't get a chance to make a protein or maybe it doesn't get to make too many copies of the protein. If it's persistent for a long time, it can make a lot of copies of protein.

All of those things can and do occur. Then, of course, there is the regulation at the level of translation.

Translation, if I give you an mRNA, is it automatically going to be translated? Maybe the cell has a way to sequester the RNA to ramp it up in some way so that it doesn't get to the ribosome under some conditions, and under other conditions it does get to the ribosome, or some ways to block in other manners than just sequestering it, but to physically block whether or not this message gets translated, what turns out that there's a tremendous amount of that. It's, again, not the most common, but we're learning, particularly over the last couple of years, that regulation of the translation of an mRNA is important.

There are, although I won't talk about them at length, an exciting new set of genes called micro RNA's, teeny little RNAs that encode 21-22 base pair segments that are able to pair with a messenger RNA and interfere in some ways partially with its translatability. And so, by the number and the kinds of little micro RNAs that are there, organisms can tweak up or down how actively a particular message is being translated.

So, the ability to regulate translation in a number of different ways is important. And then, of course, there's post-translational control. Once a protein is made, there's post-translational regulation that could happen.

It could be that the protein is modified in some way.

The proteins say completely inactive unless you put a phosphate group on it, and some enzyme comes along and puts a phosphate group on it. Or, it's inactive until you take off the phosphate group.

All sorts of post-translational modifications can occur to proteins after the amino acid chain is made that can affect whether or not the protein is active. Every one of these is potentially a step by which an organism can regulate whether or not you have a certain biochemical activity present in a certain amount at a certain time. And, every one of these gets used. This is the thing about coming to a system that has been in the process of evolution for three and a half billion years is that even little differences can be fought over as competitive advantages, and can be fixed by an organism. So, if a tiny little thing began to help the organism slightly, it could reach fixation. And, you're coming along to this system, which has had about three and a half billion years of patches to the software code, and it's just got all sorts of layers and regulation piled on top of it. All of these things happen. But, what we think is the most important out of this whole collection is this guy.

The fundamental place at which you're going to regulate whether or not you have the product of a gene is whether you bother to transcribe its RNA. But I do want to say because, yes? And, which exons you used and which aren't? Yeah, well, there are tissue-specific factors that are gene-specific that can influence that. And, surprisingly little is known about the details. There are a couple of cases where people know, but as you'd imagine, you actually need a regulatory system in that tissue to be able to decide to skip over that exon.

And, the mechanics of that surprisingly are understood in very few cases. And, you might think that evolution wouldn't like to use that as the most common thing because you really do have to make a specialized thing to do that. So, that's what happens on these. That's one in particular where I think a tremendous amount of more work has to happen.

mRNA stability, we understand some of it but not all the factors in this business. I was telling you about translation with these little micro-RNAs is stuff that's really only a few years old that people have come to understand. So, there's a lot to be understood about these things. I'm going to tell you about initiation of mRNAs, because it's the area where we know the most, and I think it'll give you a good idea of the general paradigm.

But, any of you who want to go into this will find that there's a tremendous amount more to still be discovered about these things.

So, the amount of protein that a cell might make varies wildly.

Your red blood cells, 80% of your red blood cells, protein, is alpha or beta-globin. It's a huge amount. That's not true in any other cell in your body. So, we were talking about pretty significant ranges of difference as to how much protein is made.

How do things like that happen? Well, I'm going to describe the simplest and classic case of gene regulation and bacteria, and in particular, the famous lack operon of E coli.

So, this was the first case in which regulation was ever really worked out, and it stands today as a very good paradigm of how regulation works. E coli, in order to grow, needs a carbon source. In particular, E coli is fond of sugar.

It would like to have a sugar to grow on. Given a choice, what's E coli's favorite sugar? It's glucose, right, because we have the whole cycle of glucose. The whole pathway of glucose goes to pyruvate, which we've talked about, and glucose is the preferred sugar to go into that pathway, OK, of glycolysis.

Glycolysis: the breakdown of glucose. But, suppose there's no glucose available. Is E coli willing to have a different sugar?

Sure, because E coli's not stupid. If it were to refuse another sugar, it wouldn't be able to grow. So, it has a variety of pathways that will shunt other sugars to glucose, which will then allow you to go through glycolysis, etc. Now, given a choice, it would prefer to use the glucose. But if not, suppose you gave it lactose. Lactose is a disaccharide. It's milk sugar, and I'll just briefly sketch, so lactose is a disaccharide where you've got a glucose and a galactose.

Glucose plus galactose equals lactose. So, if E coli is given galactose, it is able to break it down into glucose plus galactose.

And it does that by a particular enzyme called beta galactosidase, which breaks down glactosides. And, it'll give you galactose plus glucose. How much beta-galactosidase does an E coli cell have around? Sorry? None? But how does it do this?

When it needs it, it'll synthesize it. When it needs it, like, there's no glucose and there's a lot of galactose around, how much of it will there be? A lot. It turns out that in circumstances where E coli is dependent on galactose as its fuel, something like 10% of total protein can be beta-gal under the circumstances when you have galactose but no glucose. Sorry? Sorry, when you have lactose but no glucose. Thank you. So, when you have lactose but no glucose, E coli has 10% of its protein weight as beta-galactosidase. Wow. But when you have glucose around or you don't have lactose around, you have very little.

It could be almost none, trace amounts. So, why do this?

Why not, for example, just have a far more reasonable some compromise?

Like, let's always just have 1% of beta-galactosidase.

Why do we need the 0-10%? 10%'s actually extremely high.

So what. It's a good insurance policy. So, if I only have galactose, I need more. Well, I mean, 1% will still digest it. I'll still do it. What's the problem? Sorry?

So what, I do it at a slower rate. Life's long. Why not? Ah, it has to compete. So, if the cell to the left had a mutation that got it to produce four times as much, then it would soak up the lactose in the environment, grow faster, etc. etc., and we could have competed.

So, these little tuning mutations have a huge effect amongst this competing population of bacteria. And so, if E coli currently thinks that it's really good to have almost non at sometimes and 10% at other times, you can bet that it's worked that out through the product of pretty rigorous competition, that it doesn't want to waste the energy making this when you don't need it, and that when you do need it, you really have to compete hard by growing as fast as you can when you have that lactose around. OK. So, how does it actually get the lactose, sorry, keep me honest on lactose versus galactose, into the cell? It turns out that it also has another gene product, another protein, which is a lactose permease. And, any guesses as to what a lactose permease does? It makes the cell permeable to lactose, right, good. So, the lactose can get into the cell, and then beta-gal can break it down into galactose plus glucose. These two things, in fact, both get regulated, beta-gal and this lactose permease. So, how does it work?

Let's take a look now at the structure of the lack operon.

So, I mentioned briefly last time, what's an operon? Remember we said that in bacteria, you often made a transcript that had multiple proteins that were encoded on it.

A single mRNA could get made, and multiple starts for translation could occur, and you could make multiple proteins.

And, this would be a good thing if you wanted to make a bunch of proteins that were a part of the same biochemical pathway.

Such an object, a regulated piece of DNA that makes a transcript encoding multiple polypeptides is called an operon because they're operated together. So, let's take a look here at the lack operon. I said there was a promoter.

Here is a promoter for the operon, and we'll call it P lack, promoter for the lack operon. Here is the first gene that is encoded. So, the message will start here, actually about here, and start going off. And, the first gene is given the name lack Z.

It happens to encode beta-galactosidase enzyme.

Remember, they did a mutant hunt, and when they did the mutant hunt, they didn't know what each gene was as they isolated mutants.

So, they just gave them names of letters. And so, it's called lack Z. And, everybody in molecular biology knows this is the lack Z gene, although Z has nothing to do with beta-galactosidase. It was just the letter given to it.

But, it's stuck. Next is lack Y.

And, that encodes the permease. And, there is also lack A, which encodes a transacetylase, and as far as I'm concerned you can forget about it. OK, but I just mentioned that it is there, and it actually does make three polypeptides.

We won't worry about it, OK, but it does make a transacetylase, OK? But it won't figure in what we're going to talk about, and actually remarkably little is known about the transacetylase. There's also one other gene I need to talk about, and that's over here, and that's called lack I. And, it too has a promoter, which we can call PI, for the promoter for lack I.

And, this encodes a very interesting protein.

So, we get here one message encoding one polypeptide here.

This mRNA encodes one polypeptide. It is monocystronic. This guy here is a polycystronic message. It has multiple cystrons, which is the dusty old name for these regions that were translated into distinct proteins. And so, that's that mRNA.

So, lack I, this encodes a very interesting protein, which is called the lack repressor. The lack repressor, actually I'll bring this down a moment, is not an enzyme.

It's not a self-surface channel for putting in galactose.

It is a DNA binding protein. It binds to DNA. But, it's not a nonspecific DNA binding protein that binds to any old DNA.

It has a sequence-specific preference.

It's a protein that has a particular confirmation, a particular shape, a particular set of amino acids sticking out, that it combined into the major groove of DNA in a sequence-specific fashion such that it particularly likes to recognize a certain sequence of nucleotides and binds there. Where is the specific sequence of nucleotides where this guy likes to bind? It so happens that it's there.

And this is called the operator sequence or the operator site.

So, this protein likes to go and bind there. Now, I've drawn this, by the way, so that this operator site is actually right overlapping the promoter site.

Who likes to bind at the promoter site? RNA polymerase.

What's going to happen if the lack repressor protein is sitting there?

RNA polymerase can't bind. It's just physically, blocked from binding. So, let's examine some cases here.

Let's suppose that we look at here at our gene. We've got our promoter, P lack. We've got the operator site here. We've got the lack Z gene here, and we've got the lack repressor, lack I, the repressor sitting there.

Polymerase tries to come along to this, and it's blocked.

So, what will happen in terms of the transcription of the lack operon: no mRNA. So, that's great.

So, we've solved one problem right off the bat.

We want to be sure that sometimes there's going to be no mRNA made.

This way, we're not going to waste any metabolic energy, making beta-galactosidase. Are we done? No? Why not.

We've got to sometimes make beta-galactosidase.

So, we've got to get that repressor off there. Well, how is the repressor going to come off there? When do we want the repressor off there: when there's lactose present.

So, somehow we need to build some kind of an elaborate sensory mechanism that is able to tell when lactose is present, and send a signal to the repressor protein saying, hey, lactose is around. The signal gets transmitted all the way to the repressor protein, and the repressor protein comes off.

What kind of an elaborate sensory mechanism might be built?

Use lactose as what? So, this is actually pretty simple.

You're saying just take lactose, and you want lactose to be its own signal? So, if lactose were to just bind to the repressor, the repressor might then know that there was lactose around.

Well, what would it do if lactose bound to it? Sorry? Why would it fall off? Yep. More interested in the lactose.

So, if you're suggestion, this is good. I like the design work going on here. The suggestion is that if lactose binds to this here, binds to our repressor, it's going to fall off because it's more interested in lactose than in the DNA. Now, how is the interest actually conveyed into something material? Because the actual level of cognitive like or dislike for DNA on the part of this polypeptide is unclear, you may be anthropomorphizing slightly with regard to this polypeptide chain. So, mechanistically, what's going to happen? Shape. Yes, shape? Change confirmation, the binding act, the act of binding lactose creates some energy, may change the shape of the protein, and that shape of the protein may, in the process of wiggling around to bind lactose may de-wiggle some other part of it that now no longer binds so well to DNA. That is exactly what happens.

Good job. So, you guys have designed, in fact, what really happens. What happens is what's called an allosteric change. It just means other shape.

So, it just changes its shape, that it changes shape on binding of lactose. And it falls off because it's less suitable for binding this particular DNA sequence when it's bound to lactose there. So, in this case, in the presence of lactose, lack I does not bind.

And, the lack operon is transcribed. Yes? Uh-oh. OK, all right designers, here we've got a problem. You have such a cool system, right? You were going to sense lactose.

Lactose was going to bind to the lack repressor, change its confirmation falloff: uh-oh. But, as you point out, how's it going to get any lactose, because there's not a lactose permease because the lactose permease is made by the same operon. So, what if, in fact, instead of getting one of these DOD mill speck kind of things of some repressor that is absolutely so tight that it never falls off under any circumstances, what if we build a slightly sloppy repressor that occasionally falls off, and occasionally allows transcription of the lack operon? Then, we'll have some trace quantities of permease around. With a little bit of permease around, a little lactose will get in.

And, as long as even a little lactose gets in, it'll now shift the equilibrium so that the repressor is off more, and of course that will make more permease, and shift, and shift, and shift, and shift. So, as long as it's not so perfectly engineered as to have nothing being transcribed, so no mRNA is really very little mRNA. See, this is what's so good, I think, about having MIT students learn this stuff because there are all sorts of wonderful design principles here about how you build systems. And, I think this is just a very good example of how you build a system like this.

Now, all right, so we now have the ability to have lack on and lack off, and that is lack off, mostly off because of your permease problem: very good. Now, let's take a little digression about, how do we know this? This kind of reasoning, I've now told you the answer. But let's actually take a look at understanding the evidence that lets you conclude this.

So, in order to do this, and this is the famous work in molecular biology of Jacobin Manoux in the late '50s for which they won a Nobel Prize, they wanted to collect some mutants.

Remember, this is before the time of DNA sequence or anything like that, and wanted to collect mutants that affected this process.

So, in order to collect mutants that screwed up the regulation, they knew that beta-galactosidase was produced in much higher quantity if lactose was around. The difficulty with that was that wild type E coli, when you had no lactose would produce very little beta-gal, one unit of beta-gal, and in the presence of lactose, would produce a lot, let's call it 1, 00 units of beta-gal. But, the problem with playing around with this is lactose is serving two different roles.

Lactose is both the inducer of the expression of the gene by virtue of binding to the repressor, etc., etc.

But, it's also the substrate for the enzyme because as beta-galactosidase gets made, it breaks down the lactose. So, there's less lactose in binding, and if you wanted to really study the regulatory controls, you have the problem that the thing that's inducing the gene by binding to the repressor is the thing that's getting destroyed by the product of the gene. So, it's going to make the kinetics of studying such a process really messy. It would be very nice if you could make a form of lactose that could induce beta-galactosidase by binding to the repressor, but wasn't itself digested.

Chemically, in fact, you can do that. Chemically, it's possible to make a molecule called IPTG, which is a galactoside analog. And, what it does is this molecule here which I'll just sketch very quickly here, it's a sulfur there, and you can see vaguely similar, this is able to be an inducer.

It'll induce beta-gal, but not a substrate. It won't get digested.

So, it'll stick around as long as you want. It's also very convenient to use a molecule that was developed called ex-gal.

Ex-gal again has a sugar moiety, and then it also has this kind of a funny double ring here, which is a chlorine, and a bromine, and etc. And, this guy here is not an inducer. It's not capable of being induced, of inducing beta-galactosidase expression. But, it is a substrate.

It will be broken down by the enzyme, and rather neatly when it's broken down it turns blue. These two chemicals turned out to be very handy in trying to work out the regulation of the lack operon. So, if I, instead of adding lactose, if I think about adding IPTG, my inducer, when I add IPTG I'm going to get beta-gal produced. When I don't have IPTG, I won't produce beta-gal. But then I don't have a problem of this getting used up. So now, what kind of a mutant might I look for? I might look for a mutant that even in the absence of the inducer, IPTG, still produces a lot of beta-gal. Now, I can also look for mutants that no matter what never produce beta-gal, right? But, what would they likely be? They'd likely be structural mutations affecting the coding sequence of beta-gal, right? Those will happen.

I can collect mutations that cause the E coli never to produce beta-gal. But that's not as interesting as collecting mutations that block the repression that cause beta-gal to be produced all of the time. So, how would I find such a mutant?

I want to find a mutant that's producing a lot of beta-gal even when there's no IPTG. So, let's place some E coli on a plate. Should we put IPTG on a plate? No, so no IPTG.

What do I look for? How do I tell whether or not any of these guys here is producing a lot of beta-gal? Yep?

So, no IPTG, but put on ex-gal, and if anybody's producing a lot of beta-gal, what happens? They turn blue: very easy to go through lots of E coli like that looking for something blue.

And so, lots of mutants were collected that were blue.

And, these chemicals are still used today. They're routinely used in labs, ex-gal and stuff like that, making bugs turn blue because this has turned out to be such a well-studied system that we use it for a lot of things. So, mutants were found that were constituative. So, mutants were found that were constituative mutants. Constituative mutants: meaning expressing all the time, no longer regulated, so, characterizing these constituative mutants.

It turns out that they fell into two different classes of constituative mutants. If we had enough time, and you could read the papers and all, what I would do is give you the descriptions that Jacobin Maneaux had of these funny mutants which they'd isolated and were trying to characterize, and how to puzzle out what was going on.

But, it's complicated and hard, and makes your head hurt if you don't know what the answer is. So, I'm going to first tell you the answer of what's going on, and then sort of see how you would know that this was the case. But, imagine that you didn't know this answer, and had to puzzle this out from the data.

So, suppose we had, so if there were going to be two kinds of mutants: mutant number one are operator constituents.

They have a defective operator sequence. Mutations have occurred at the operator site. Mutant number two have a defective repressor protein, the gene for the repressor protein.

How can I tell the difference?

So, I could have a problem in my operator site.

What would be the problem with the operator site?

Some mutation to the sequence causes the repressor not to bind there anymore, OK? So, a defective operator site doesn't bind repressors. Defective repressor, the operator site is just fine, but I don't have a repressor to bind at it. So how do I tell the difference? One way to tell the difference is to begin crossing the mutants together to wild type, and asking, are they dominant or recessive, or things like that?

Now, here's a little problem. E Coli is not a diploid, so you can't cross together two E colis and make a diploid E coli, right? It's a prokaryote. It only has one genome. But, it turns out that you can make temporary diploids, partial diploids out of E coli because it turns out you can mate bacteria. Bacteria, which have a bacterial chromosome here also engage in sex and in the course of bacterial sex, plasmids can be transferred called, for example, an F factor, is able to be transferred from another bacteria. And, through the wonders of partial merodiploid, you can temporarily get E colis, or you can permanently get E colis, that are partially diploid. So, you can do what I'm about to say. But, in case you were worried about my writing diploid genotypes for E coli, you can actually do this.

You can make partial diploids. So, let's try out a genotype here.

Suppose the repressor is a wild type, the operator is wild type, and the lack Z gene is wild type. And, suppose I have no IPTG, I'm un-induced. I have one unit of beta-gal. When I add my inducer, what happens? I get 1,000 units of beta-gal.

Now, suppose I would have an operator constituative mutation.

Then, the operator site is defective. It doesn't bind the repressor. Beta-gal is going to be expressed all the time, even in the absence. All right, well that was, of course, what we selected for. Now, suppose I made the following diploid.

I plus, O plus, Z plus, over I plus, O constituative, Z plus. So, here's my diploid. What would be the phenotype? So, in other words, one of the chromosomes has an operator problem.

Well, that means that this chromosome here is always going to be constituatively expressing beta-gal.

But, what about this chromosome here? It won't. So, this would be about 1, 01, give or take, because it's got one chromosome doing that and one chromosome doing this, and this one would be about 2, 00. Now, that quantitative difference doesn't matter a lot. What you really saw when you did the molecular biology was that when you had one copy of the operator constituative mutation, you still got a lot of beta-gal here even in the absence of IPTG. So, that operator constituative site looked like it was dominant to this plus site here.

But now, let's try this one here. I plus, O plus, Z plus, over I plus, operator constituative, Z minus. What happens then?

This operator constituative site allows constant transcription of this particular copy. But, can this particular copy make a working, functional beta-gal? No. So, this looks, when you do your genetic crosses, you find that the operator constituative, now, if I reverse these here, suppose I reverse these, I plus, O plus, Z minus, I plus, O constituative, Z plus, same genotypes, right, except that I flipped which chromosome these are on.

Now, what happens? This chromosome here: always making beta-gal and it works. This chromosome here: not making beta-gal.

Even though it's regulated, it's a mutant. So, in other words, from this very experiment, you can tell that the operator site is only affecting the chromosome that it's physically on, that it doesn't make a protein that floats around.

What it does is it's said to work in cys. In cys means on the same chromosome. It physically works on the same chromosome.

Now, let's take a look, by contrast, of the properties of the lack repressor mutants. If I give you a lack repressor mutant, I plus, O plus, Z plus is the wild type.

I constituative, O plus, Z plus: what happens here?

This wild type is one in 1, 00. This guy here: 1,000 and 1, 00, and then here let's look at a diploid: I plus, O plus, Z plus, I constituative, O plus, Z plus. What's the effect? The I constituative doesn't make a functioning repressor. But, I plus makes a functioning repressor. So, will this show regulation?

Yeah, this will be regulated just fine. This works out just fine, and in fact it'll make 2,000, and it'll make two copies there.

But again, the units don't matter too much. And, by contrast, if I give you I plus, O plus, Z minus, and I constituative, O plus, Z plus, what will happen?

Here, I have my mutation on this chromosome. But, it doesn't matter because I've got my mutation on this chromosome in the repressor. I've got a mutation on lack Z here, but as long as I have a functional copy, one functional copy of the lack repressor, it works on both chromosomes.

It will work on both chromosomes, and so in other words this lack repressor, one copy works on both chromosomes. In other words, it makes a product that diffuses around, and can work on either chromosome, and it's said to work in trans, that is, across.

So, the operator is working in cys. It's operating on its own chromosome only. A mutation in the operator only affects the chromosome it lives on, whereas a functional copy of the lack repressor will float around because it's a protein, and that's how Jacobin Maneaux knew the difference.

They proved their model by showing that these two kinds of mutations had very different properties. Operator mutations affected only the physical chromosome on which they occurred, which of course they had to infer from the genetics they did, whereas repressor, a functional copy repressor, could act on any chromosome in the cell.

So, OK, we've got that. Now, last point, what about glucose?

I haven't said a word about glucose. See, this was a big deal to people.

This model, the repressor model, we have this repressor. What about glucose? What's glucose doing in this picture?

So, glucose control: so here's my gene. Here's my promoter, P lack. Here's my operator, beta-gal.

It's encoded by lack Z. You've got all that. When this guy is present, sorry, when lactose is present, the repressor comes off. Polymerase sits down. Wait a second, polymerase isn't supposed to sit down unless there's no glucose.

We need another sensor to tell if there's glucose, or if there's low glucose. So, we're going to need us a sensor that tells that. Any ideas? Yep?

Yeah, if you work that one through, I don't think it quite works. But, you've got the basic idea. You're going to want another something, and it turns out there's another site over here, OK? There's a second site on which a completely different protein binds. And, this protein is the cyclic AMP regulatory protein, and it so happens that in the cell, when there's low amounts of glucose, let me make sure I've got this right, when there's low amounts of glucose, what we have is high amounts of cyclic AMP. Cyclic AMP turns out, whereas lactose is used directly as the signal, cyclic AMP is used as the signal here. When the cell has low amounts of glucose, it has high amounts of cyclic AMP. Now, what do you want your cyclic AMP to do? How are we going to design this?

It's going to bind to a protein, cyclic AMP regulatory protein, it's going to sit down, and now what's it going to do?

Is it going to block RNA polymerase?

What do we want to do? If there's low glucose, high cyclic AMP, we sit down at the site, we want to turn on transcription now, right? So, what it's got to do is not block RNA polymerase, but help RNA polymerase. So, what it actually does is instead of being a repressor, it's an activator. And what it does is it makes it more attractive for RNA polymerase to bind, and it actually does that by, actually it does it slightly by bending the DNA.

But, what it does is it makes it easier for RNA polymerase to bind.

It turns out that the promoter is kind of a crummy promoter.

It's actually just like, remember the repressor wasn't perfect the promoter's not perfect either. The promoter's kind of crummy.

And, unless RNA polymerase gets a little help from this other regulatory protein, it doesn't work.

We have two controls: a negative regulator responding to an environmental cue, a positive activator responding to an environmental cue, helping polymerase decide whether to transcribe or not, and basically that's how a human egg goes to a complete adult and lives its entire life, minus a few other details. There are some details left out, but that's a sketch of how you turn genes on and off.


Post Translational Modifications: An Overview

Cells need to detect and react to changes in internal and external conditions . One method used to adjust to these changes is chemically modifying proteins. Conditional chemical changes are relayed from sensors to effectors via reversible post-translational modifications (PTMs) of proteins. PTMs play an important part in modifying the end product of expression, contribute to biological processes and diseased conditions, playing a key role in many cellular processes such as cellular differentiation (1), protein degradation, signaling and regulatory processes, regulation of gene expression, and protein-protein interactions (2,3).

How does post translational modification work?

PTMs can happen at any step of the protein lifespan. Many proteins are modified shortly after translation is completed to mediate proper folding or to direct the nascent protein to distinct cellular locations (such as the nucleus or membrane). Other modifications occur after folding and localization are completed to activate or inactivate catalytic activity. Proteins are also covalently linked to tags that target a protein for degradation. They are modified through a combination of post-translational cleavage and the addition of functional groups through a step-wise mechanism of protein maturation or activation.

Where does post translational modification occur? PTMs occur at distinct amino acid side chains or peptide linkages and are most often mediated by enzymatic activity. Indeed, 5% of the proteome comprises enzymes that perform more than 200 types of PTMs (4). These enzymes include kinases, phosphatases, transferases, and ligases, which add or remove functional groups, proteins, lipids, or sugars to or from amino acid side chains, and proteases, which cleave peptide bonds to remove specific sequences or regulatory subunits. Many proteins can also modify themselves using autocatalytic domains, such as autokinase and autoprotolytic domains. PTMs can also be reversible based on the nature of the modification. As an example, phosphatases hydrolyze the phosphate group to remove it from the protein and reverse its biological activity (Figure 1).

Figure 1. Types of post-translational modifications (PTMs).

Most common post-translational modifications

Recent developments in mass-spectrometry (MS) methods have enabled the identification of thousands of PTM sites. Consequently, novel enrichment strategies have uncovered the global cellular importance of several types of modifications (e.g., acetylation, ubiquitylation, O-GlNac, N-linked glycosylation). More than 200 diverse types of PTMs are currently known (5,6), ranging from small chemical modifications (e.g., phosphorylation and acetylation) to the addition of complete proteins (e.g., ubiquitylation, Figure 3).

Phosphorylation

Protein phosphorylation (Figure 2) is the most commonly studied post-translational modification. It has been estimated that one-third of mammalian proteins may be phosphorylated, and this modification often plays a key role in modulating protein function. Phosphorylation takes place on serine, threonine, and tyrosine residues, acting to regulate protein function, enzymatic activity, protein–protein interactions, and protein localization. Phosphorylation is catalyzed by phosphatases and can be reversible – phosphorylated proteins can be dephosphorylated by protein dephosphatases.

Figure 2. WB result of phospho-Marcks antibody ( 10018-3-AP , 1:1500) with mouse J774 macrophage cells treated with PMA.

Glycosylation and Glycanation

The majority of proteins that are synthetized on ribosomes associated with the endoplasmic reticulum undergo glycosylation. That means a covalent attachment of sugar moieties is added to the polypeptide chain. The two most common types of glycosylation in Eukaryotes are N-linked glycosylation – to asparagine, and O-linked glycosylation – to serine and threonine.

Ubiquitination

Protein ubiquitination means a covalent ubiquitin is added to lysine, cysteine, serine, threonine, or directly to the protein N-terminus. Ubiquitin is a small (+/-8.6 kDa) protein expressed across almost all tissue types (Figure 3). Ubiquitination is an enzymatic reaction catalyzed by a three-enzyme cascade (E1, E2, and E3). That provides substrate specificity and activation, conjugation, and ligation steps. Proteins can be monoubiquitinated (with one ubiquitin molecule) or polyubiquitinated. Polyubiquitination takes place when additional ubiquitin molecules are added to the initial ubiquitin molecule. Ubiquitination via the proteome can mark proteins for degradation. It is also important for cellular signaling, the internalization of membrane proteins , and the development and regulation of transcription.

Figure 3. MDA-MB-453s cells were subjected to SDS PAGE followed by western blot with 10201-2-AP (ubiquitin antibody) at a dilution of 1:600.

PTMs impact on health and disease

The analysis of proteins and their PTMs is particularly important for the study of heart disease, cancer, neurodegenerative diseases, and diabetes (7). The main challenges in studying post-translationally modified proteins are the development of specific detection and purification methods. Fortunately, these technical obstacles are being overcome with a variety of new and refined proteomics technologies.


Multitasking the proteome

Another feature of PTMs is that they fulfil a multitude of different functions in proteins that harbour them. The addition or removal of PTMs regulates enzyme activity, intra- and intermolecular changes in protein conformation, cellular localization, and interactions with protein partners or other biomolecules. Extraordinarily, a single type of PTM can be responsible for several of these different functions, depending on the site of modification and the context of the signalling pathway. This is particularly well exemplified by ubiquitin signalling, in which the highly conserved small polypeptide ubiquitin is attached to lysine residues of target proteins. Proteins may be modified by a single ubiquitin polypeptide (i.e. monoubiquitination) or at multiple different positions (multi-monoubiquitination). Moreover, seven internal lysine residues allow ubiquitin to form homogenous or heterogenous chains that in some cases are branched. These vastly different forms of ubiquitination allow this PTM to signal for a wide variety of processes that range from cellular signalling and protein sorting to targeted proteolysis ( Komander and Rape, 2012). Miricescu et al. (2018) review how the multi-functionality of ubiquitin integrates environmental signals, illustrating that this PTM in all its different forms plays a central role in nearly all pathways regulated by developmental and stress hormones. Emerging evidence also indicates that ubiquitin modifications orchestrate cross talk between synergistic and antagonistic hormone-signalling pathways, allowing plants to fine tune cellular responses to their environment. The complexity provided by this PTM alone may explain why so much of each plant genome is devoted to encoding genes related to ubiquitin signalling.

Plant hormone perception represents a particularly intriguing multifunctional role for ubiquitin in the reprogramming of gene expression. Both Adams and Spoel (2018) and Miricescu et al. (2018) discuss how several plant hormones are perceived at the chromatin by ubiquitin E3 ligases. These E3 ligases multitask by functioning as direct hormone receptors and simultaneously as transcriptional cofactors. Multitasking is enabled by hormones acting as a molecular glue between the E3 ligase and its substrate, which is either a transcriptional coactivator or corepressor. The resulting hormone-induced ubiquitination of transcriptional co-regulators alters their intrinsic properties or targets them for degradation ( Kelley and Estelle, 2012 Furniss and Spoel, 2015). Notably, elucidating the mechanisms by which these chromatin-associated E3 ligases multitask has far-reaching implications beyond plant biology. The biomedical sciences have vested interests in the design of therapeutic agents that mimic small molecule perception by E3 ligases, as dysfunction in ubiquitin signalling underpins many pathophysiological conditions in humans, including cancer, neurological disorders and immunodeficiency. Consequently, pharmacological drug discovery approaches are now being inspired by the mechanisms of plant hormone perception ( Shabek and Zheng, 2014). By studying this natural process in plants we can contribute in unexpected ways to advances in biomedicine.


Proteomics in Biology, Part B

C. Zhang , Y. Liu , in Methods in Enzymology , 2017

Abstract

Posttranslational modifications (PTMs) of histones are one of the main research interests in the rapidly growing field of epigenetics. Accurate and precise quantification of these highly complex histone PTMs is critical for understanding the histone code and the biological significance behind it. It nonetheless remains a major analytical challenge. Mass spectrometry (MS) has been proven as a robust tool in retrieving quantitative information of histone PTMs, and a variety of MS-based quantitative strategies have been successfully developed and employed in basic research as well as clinical studies. In this chapter, we provide an overview for quantitative analysis of histone PTMs, often highly flexible and case dependent, as a primer for future experimental designs.


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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

AGOargonaute protein
ALPautophagy lysosomal pathway
ALSamyotrophic lateral sclerosis
AMPKAMP-activated protein kinase
ARE-RBPRNA-binding proteins that recognize AU-rich elements
AREAU-rich element
ATMataxia-telangiectasia mutated
CARM1coactivator-associated arginine methyltransferase 1
CBPcAMP-response element binding protein (CREB)-binding protein
CD62EE-selectin
Chk2checkpoint kinase 2
Clk1Cdc-like kinase 1
COX-2cyclooxygenase 2
CSEcystathionine γ-lyase
CTSScathepsin S
CUREsCU-rich elements
DDRDNA damage response
DGCR8DiGeorge syndrome critical region 8
DICEdifferentiation control element
ECRG2esophageal cancer-related gene 2
eNOSendothelial nitric oxide synthase
ERKextracellular signal-regulated kinase
EV71enterovirus 71
FASTKFas-activated serine/threonine kinase
FBXW2F-box/WD repeat-containing protein 2
FTLDfronto-temporal lobar degeneration
FUSfused in sarcoma
FXR2Pfragile X-related protein 2
GM-CSFgranulocyte-macrophage colony-stimulating factor
GSK3 βglycogen synthase kinase 3 β
G3BP1Ras-GAP SH3 domain-binding protein 1
HDM2/MDM2human/mouse double minute 2
hnRNP A1/A2/Kheterogeneous nuclear ribonucleoprotein A1 A2 and K
HNSHuR nucleocytoplasmic shuttling sequence
Hsp27heat shock protein 27
HuRhuman antigen R
IDRintrinsically disordered region
IKK αI κ B kinase α
IRESinternal ribosome entry site
JAK3Janus kinase 3
KHK homology
KIK interactive region
KLF2Krüppel-like factor 2
KNSK nuclear shuttling domain
KSRPKH-type splicing regulatory protein
LCRlow-complexity region
LLPSliquid–liquid phase separation
MAPKmitogen-activated protein kinase
MAPKAPK-2/3MAPK-activated protein kinase 2 and 3
mFasmembrane-bound Fas
miRISCmiRNA-induced silencing complex
miRNAmicro-RNA
MK2/3MAPK-activated protein kinase 2 and 3
MLOmembrane-less organelle
MMP2/7matrix metalloproteinase 2 and 7
mRNPmessenger ribonucleoprotein particle
NCLnucleolin
NEDD8neural precursor cell expressed developmentally downregulated 8
NLSnuclear localization signal
O-GlcNAcO-glycosyl -N-acetylation
PABPpoly(A)-binding protein
PAD4peptidyl arginine deiminase 4
PARpoly(ADP-ribose)
PARP-1PAR polymerase 1
Pc2polycomb 2 protein
Pin1protein interacting with NIMA (never in mitosis A)-1
PKAprotein kinase A
PKC α/δ/ζprotein kinase C α δ and ζ
PLDprion-like domain
pre-miRNAprecursor miRNA
pri-miRNAprimary miRNA
PRMT1protein arginine N-methyltransferase 1
PTHparathyroid hormone
PTMpost-translational modification
r15-LOXerythroid-15-lipoxygenase
RBDRNA-binding domain
RBPRNA-binding protein
RGG/RGarginine/glycine-rich
RRMRNA recognition motif
sFassoluble Fas
SGsstress granules
SIRT1Sirtuin 1
SMNsurvival of motor neuron
snRNP U1small nuclear ribonucleoprotein particle U1
SRserine/arginine-rich
SRPK1/2SR protein kinase 1 and 2
SRSF1/3SR splicing factor 1 and 3
StUbLSUMO-targeted ubiquitin ligase
SUMOsmall ubiquitin-like modifier
TDP-43TAR DNA-binding protein of 43 kDa
TIA-1T-cell intracellular antigen 1
TIARTIA-1-related
TIARTIA-1-related protein
TRAF6TNF receptor-associated factor 6
TRNtransportin
TTPtristetraprolin
Ububiquitin
UBXD8ubiquitin regulatory X domain-containing protein 8
UCP2uncoupling protein-2
UPSubiquitin-proteasome system
UTRuntranslated region
XIAPX chromosome-linked inhibitor of apoptosis protein
β-TrCP 1β-transducin repeat-containing protein 1.

Initiation

During translation, a small ribosomal subunit attaches to a mRNA molecule. At the same time an initiator tRNA molecule recognizes and binds to a specific codon sequence on the same mRNA molecule. A large ribosomal subunit then joins the newly formed complex. The initiator tRNA resides in one binding site of the ribosome called the P site, leaving the second binding site, the A site, open. When a new tRNA molecule recognizes the next codon sequence on the mRNA, it attaches to the open A site. A peptide bond forms connecting the amino acid of the tRNA in the P site to the amino acid of the tRNA in the A binding site.


Websites

Teachers' Domain: Cell Transcription and Translation

Teachers' Domain is a free educational resource produced by WGBH with funding from the NSF, which houses thousands of media resources, support materials, and tools for classroom lessons.One of these resources focuses on the topics of transcription and translation.This resource is an interactive activity that starts with a general overview of the central dogma of molecular biology, and then goes into more specific details about the processes of transcription and translation.In addition to the interactive activity, the resource also includes a background narrative and discussion questions that could be used for assessment.Although the material is designated as appropriate content for grades, 9-12, it would serve as an excellent introduction to the topic for biology majors, or would be well suited for non-biology majors at the post-secondary level. See: Teachers' Domain: Cell Transcription and Translation

The DNA Learning Center's (DNALC) The Howard Hughes Medical Institute's DNA interactive (DNAi) The University of Utah's Genetic Science Learning Center

The DNA Learning Center's (DNALC) website, the Howard Hughes Medical Institute's DNA interactive (DNAi) website, and the University of Utah's Genetic Science Learning Center website listed below contain excellent narrated animations describing transcription and translation. These animations are useful as a lecture supplement or for students to review on their own. The DNALC animations cover central dogma, transcription (basic and advanced), mRNA splicing, RNA splicing, triplet code and translation (basic and advanced). The DNAi modules," Reading the Code" and "Copying the Code," describe the history of the process, the scientists involved in the discovery, and the basics of the process, and also include an animation and interactive game. Particularly useful to students are the interactive animations from the University of Utah that allow one to, for example,"Transcribe/Translate a Gene"or examine the effects of gene mutation as they "Test Neurofibromin Activity in a Cell."

The DNA Learning Center's (DNALC): 3-D Animation Library
The Howard Hughes Medical Institute's DNA interactive: (DNAi): Code
The University of Utah's Genetic Science Learning Center: Transcribe and Translate a Gene

Scitable

The Nature Education website, Scitable, is a great study resource for students who want to learn more about, or are having difficulty understanding, transcription and translation. The site contains a searchable library, including many "overviews" of transcription, translation, and related topics. Students have access to a Genetics "Study Pack", which provides explanations, animations, and links to other resources.In addition, Scitable has an "Ask An Expert" feature that allows students to submit specific genetics-related questions. See: Scitable

NHGRI Talking Glossary of Genetics Terms iPhone App and Website

The Talking Glossary of Genetics Terms website and iPhone app provide an easily transportable and accessible reference for your students. Many times the unfamiliar vocabulary is the major stumbling block to student comprehension. This app/site gives them a handy reference to common terms used in describing the components involved on transcription and translation.
Talking Glossary of Genetics Terms
Talking Glossary of Genetics Terms iPhone App

University of Buffalo Case Study Collection: Decoding the Flu

This "clicker case" was designed to develop students' ability to read and interpret information stored in DNA. Making use of personal response systems ("clickers") along with a PowerPoint presentation, students follow the story of "Jason," a student intern at the Centers for Disease Control & Prevention (CDC). While working with a CDC team in Mexico, Jason is the only person who does not get sick from a new strain of flu. It is up to Jason to use molecular data collected from different local strains of flu to identify which one may be causing the illness. Although designed for an introductory biology course for science or non-science majors, the case could be adapted for upper-level courses by including more complex problems and aspects of gene expression, such as the excision of introns."
See: Decoding the Flu

Protein Synthesis Animation from Biology-Forums.com

Translation is the process of producing proteins from the mRNA. This YouTube video shows the molecular components involved in the process. It also animates how the peptide is elongated through interaction between mRNA, ribosome, tRNA, and residues. Protein Synthese Animation

The Central Dogma Animation by RIKEN Omics Science Center

The 'Central Dogma' of molecular biology is that 'DNA makes RNA makes protein'. This anime shows how molecular machines transcribe the genes in the DNA of every cell into portable RNA messages, how those messenger RNA are modified and exported from the nucleus, and finally how the RNA code is read to build proteins. Animation: The Central Dogma

A Prezi of this information can be found at: NHGRI Teacher Resouces-Central Dogma

Contributing Team of Educators:

Kari D. Loomis, Ph.D., Mars Hill College
Luisel Ricks, Ph.D., Howard University
Mark Bolt, Ph.D., University of Pikeville
Cathy Dobbs, Ph.D., Joliet Junior College
Changhui Yan, Ph.D., North Dakota State University
Solomon Adekunle, Ph.D., Southern University

Teachers' Domain: Cell Transcription and Translation

Teachers' Domain is a free educational resource produced by WGBH with funding from the NSF, which houses thousands of media resources, support materials, and tools for classroom lessons.One of these resources focuses on the topics of transcription and translation.This resource is an interactive activity that starts with a general overview of the central dogma of molecular biology, and then goes into more specific details about the processes of transcription and translation.In addition to the interactive activity, the resource also includes a background narrative and discussion questions that could be used for assessment.Although the material is designated as appropriate content for grades, 9-12, it would serve as an excellent introduction to the topic for biology majors, or would be well suited for non-biology majors at the post-secondary level. See: Teachers' Domain: Cell Transcription and Translation

The DNA Learning Center's (DNALC) The Howard Hughes Medical Institute's DNA interactive (DNAi) The University of Utah's Genetic Science Learning Center

The DNA Learning Center's (DNALC) website, the Howard Hughes Medical Institute's DNA interactive (DNAi) website, and the University of Utah's Genetic Science Learning Center website listed below contain excellent narrated animations describing transcription and translation. These animations are useful as a lecture supplement or for students to review on their own. The DNALC animations cover central dogma, transcription (basic and advanced), mRNA splicing, RNA splicing, triplet code and translation (basic and advanced). The DNAi modules," Reading the Code" and "Copying the Code," describe the history of the process, the scientists involved in the discovery, and the basics of the process, and also include an animation and interactive game. Particularly useful to students are the interactive animations from the University of Utah that allow one to, for example,"Transcribe/Translate a Gene"or examine the effects of gene mutation as they "Test Neurofibromin Activity in a Cell."

The DNA Learning Center's (DNALC): 3-D Animation Library
The Howard Hughes Medical Institute's DNA interactive: (DNAi): Code
The University of Utah's Genetic Science Learning Center: Transcribe and Translate a Gene

Scitable

The Nature Education website, Scitable, is a great study resource for students who want to learn more about, or are having difficulty understanding, transcription and translation. The site contains a searchable library, including many "overviews" of transcription, translation, and related topics. Students have access to a Genetics "Study Pack", which provides explanations, animations, and links to other resources.In addition, Scitable has an "Ask An Expert" feature that allows students to submit specific genetics-related questions. See: Scitable

NHGRI Talking Glossary of Genetics Terms iPhone App and Website

The Talking Glossary of Genetics Terms website and iPhone app provide an easily transportable and accessible reference for your students. Many times the unfamiliar vocabulary is the major stumbling block to student comprehension. This app/site gives them a handy reference to common terms used in describing the components involved on transcription and translation.
Talking Glossary of Genetics Terms
Talking Glossary of Genetics Terms iPhone App

University of Buffalo Case Study Collection: Decoding the Flu

This "clicker case" was designed to develop students' ability to read and interpret information stored in DNA. Making use of personal response systems ("clickers") along with a PowerPoint presentation, students follow the story of "Jason," a student intern at the Centers for Disease Control & Prevention (CDC). While working with a CDC team in Mexico, Jason is the only person who does not get sick from a new strain of flu. It is up to Jason to use molecular data collected from different local strains of flu to identify which one may be causing the illness. Although designed for an introductory biology course for science or non-science majors, the case could be adapted for upper-level courses by including more complex problems and aspects of gene expression, such as the excision of introns."
See: Decoding the Flu

Protein Synthesis Animation from Biology-Forums.com

Translation is the process of producing proteins from the mRNA. This YouTube video shows the molecular components involved in the process. It also animates how the peptide is elongated through interaction between mRNA, ribosome, tRNA, and residues. Protein Synthese Animation

The Central Dogma Animation by RIKEN Omics Science Center

The 'Central Dogma' of molecular biology is that 'DNA makes RNA makes protein'. This anime shows how molecular machines transcribe the genes in the DNA of every cell into portable RNA messages, how those messenger RNA are modified and exported from the nucleus, and finally how the RNA code is read to build proteins. Animation: The Central Dogma

A Prezi of this information can be found at: NHGRI Teacher Resouces-Central Dogma


Regulatory Proteins

Transcription regulation is mediated in part by regulatory proteins that can bind to the DNA in its helical form (unwinding is not necessary) and regulate the transcriptional activity of RNA polymerase.

Overview of regulatory proteins

  • Regulate transcription by binding to regulatory sequences of the DNA:
    • Promoters
    • Enhancer sequences
    • Insulator sequences
    • DNA binding domain: portion of the protein that binds to the DNA
    • Functional domain: portion of the protein that interacts with the DNA and/or other proteins to carry out its function
    • Helix–turn–helix
    • Zinc fingers
    • Leucine zippers

    Helix–turn–helix

    Regulatory proteins with the helix–turn–helix DNA binding motif have the following characteristics:

    • Structure: 2 helical segments oriented perpendicular to one another and connected to each other via a looping segment of protein
    • Looping segment contains the functional domain
    • Proteins bind the DNA through the major groove
    • Often work in pairs
    • A subset of helix–turn–helix proteins are known as homeodomain proteins, which are regulatory proteins often involved in development.

    2 helix–turn–helix regulatory proteins bound to DNA in the major groove

    Zinc finger

    • 3 finger-like structures interact with DNA through the major groove.
    • Uses zinc to closely associate with the DNA
    • This same motif is seen in steroid receptors, which contain a zinc-finger domain → allows the activated receptors to bind directly to the DNA and directly affect transcription

    The zinc-finger binding motif:
    Often used in steroid receptor binding mechanisms

    Leucine zipper

    Leucine zippers consist of 2 proteins, each containing a helical subunit and a hydrophobic subunit.

    • The helical subunits:
      • Enter the DNA through the major groove
      • Associate with opposite ends of the DNA within the groove
      • Remain outside the DNA (because DNA is hydrophilic)
      • Contain leucine molecules that “zip” the two proteins together

      The leucine-zipper binding motif:
      Has 2 subunits that zip together

      Related videos


      We often think of proteins as nutrients in the food we eat or the main component of muscles, but proteins are also microscopic molecules inside of cells that perform diverse and vital jobs. With the Human Genome Project complete, scientists are turning their attention to the human “proteome,” the catalog of all human proteins. This work has shown that the world of proteins is a fascinating one, full of molecules with such intricate shapes and precise functions that they seem almost fanciful.

      A protein’s function depends on its shape, and when protein formation goes awry, the resulting misshapen proteins cause problems that range from bad, when proteins neglect their important work, to ugly, when they form a sticky, clumpy mess inside of cells. Current research suggests that the world of proteins is far from pristine. Protein formation is an error-prone process, and mistakes along the way have been linked to a number of human diseases.

      The wide world of proteins:

      There are 20,000 to over 100,000 unique types of proteins within a typical human cell. Why so many? Proteins are the workhorses of the cell. Each expertly performs a specific task. Some are structural, lending stiffness and rigidity to muscle cells or long thin neurons, for example. Others bind to specific molecules and shuttle them to new locations, and still others catalyze reactions that allow cells to divide and grow. This wealth of diversity and specificity in function is made possible by a seemingly simple property of proteins: they fold.

      Proteins fold into a functional shape

      A protein starts off in the cell as a long chain of, on average, 300 building blocks called amino acids. There are 22 different types of amino acids, and their ordering determines how the protein chain will fold upon itself. When folding, two types of structures usually form first. Some regions of the protein chain coil up into slinky-like formations called “alpha helices,” while other regions fold into zigzag patterns called “beta sheets,” which resemble the folds of a paper fan. These two structures can interact to form more complex structures. For example, in one protein structure, several beta sheets wrap around themselves to form a hollow tube with a few alpha helices jutting out from one end. The tube is short and squat such that the overall structure resembles snakes (alpha helices) emerging from a can (beta sheet tube). A few other protein structures with descriptive names include the “beta barrel,” the “beta propeller,” the “alpha/beta horseshoe,” and the “jelly-roll fold.”

      These complex structures allow proteins to perform their diverse jobs in the cell. The “snakes in a can” protein, when embedded in a cell membrane, creates a tunnel that allows traffic into and out of cells. Other proteins form shapes with pockets called “active sites” that are perfectly shaped to bind to a particular molecule, like a lock and key. By folding into distinct shapes, proteins can perform very different roles despite being composed of the same basic building blocks. To draw an analogy, all vehicles are made from steel, but a racecar’s sleek shape wins races, while a bus, dump truck, crane, or zamboni are each shaped to perform their own unique tasks.

      Why does protein folding sometimes fail?

      Folding allows a protein to adopt a functional shape, but it is a complex process that sometimes fails. Protein folding can go wrong for three major reasons:

      1: A person might possess a mutation that changes an amino acid in the protein chain, making it difficult for a particular protein to find its preferred fold or “native” state. This is the case for inherited mutations, for example, those leading to cystic fibrosis or sickle cell anemia. These mutations are located in the DNA sequence or “gene” that encodes one particular protein. Therefore, these types of inherited mutations affect only that particular protein and its related function.

      2: On the other hand, protein folding failure can be viewed as an ongoing and more general process that affects many proteins. When proteins are created, the machine that reads the directions from DNA to create the long chains of amino acids can make mistakes. Scientists estimate that this machine, the ribosome, makes mistakes in as many as 1 in every 7 proteins! These mistakes can make the resulting proteins less likely to fold properly.

      3: Even if an amino acid chain has no mutations or mistakes, it may still not reach its preferred folded shape simply because proteins do not fold correctly 100% of the time. Protein folding becomes even more difficult if the conditions in the cell, like acidity and temperature, change from those to which the organism is accustomed.

      A failure in protein folding causes several known diseases, and scientists hypothesize that many more diseases may be related to folding problems. There are two completely different problems that occur in cells when their proteins do not fold properly.

      One type of problem, called “loss of function,” results when not enough of a particular protein folds properly, causing a shortage of “specialized workers” needed to do a specific job. For example, imagine that a properly folded protein is perfectly shaped to bind a toxin and break it into less toxic byproducts. Without enough of the properly folded protein available, the toxin will build up to damaging levels. As another example, a protein may be responsible for metabolizing sugar so that the cell can use it for energy. The cell will grow slowly due to lack of energy if not enough of the protein is present in its functional state. The reason the cell gets sick, in these cases, is due to a lack of one specific, properly folded, functional protein. Cystic fibrosis, Tay-Sachs disease, Marfan syndrome, and some forms of cancer are examples of diseases that result when one type of protein is not able to perform its job. Who knew that one type of protein among tens of thousands could be so important?

      Proteins that fold improperly may also impact the health of the cell regardless of the function of the protein. When proteins fail to fold into their functional state, the resulting misfolded proteins can be contorted into shapes that are unfavorable to the crowded cellular environment. Most proteins possess sticky, “water-hating” amino acids that they bury deep inside their core. Misfolded proteins wear these inner parts on the outside, like a chocolate-covered candy that has been crushed to reveal a gooey caramel center. These misfolded proteins often stick together forming clumps called “aggregates.” Scientists hypothesize that the accumulation of misfolded proteins plays a role in several neurological diseases, including Alzheimer’s, Parkinson’s, Huntington’s, and Lou Gehrig’s (ALS) disease, but scientists are still working to discover exactly how these misfolded, sticky molecules inflict their damage on cells.

      One misfolded protein stands out among the rest to deserve special attention. The “prion” protein in Creutzfeldt-Jakob disease, also known as mad cow disease, is an example of a misfolded protein gone rogue. This protein is not only irreversibly misfolded, but it converts other functional proteins into its twisted state.

      How do our cells protect themselves from misfolded proteins?

      Recent research shows that protein misfolding happens frequently inside of cells. Fortunately, cells are accustomed to coping with this problem and have several systems in place to refold or destroy aberrant protein formations.

      Chaperones are one such system. Appropriately named, they accompany proteins through the folding process, improving a protein’s chances of folding properly and even allowing some misfolded proteins the opportunity to refold. Interestingly, chaperones are proteins themselves! There are many different types of chaperones. Some cater specifically to helping one type of protein fold, while others act more generally. Some chaperones are shaped like large hollow chambers and provide proteins with a safe space, isolated from other molecules, in which to fold. Production of several chaperones is boosted when a cell encounters high temperatures or other conditions making protein folding more difficult, thus earning these chaperones the alias, “heat shock proteins.”

      Another line of cell defense against misfolded proteins is called the proteasome. If misfolded proteins linger in the cell, they will be targeted for destruction by this machine, which chews up proteins and spits them out as small fragments of amino acids. The proteasome is like a recycling center, allowing the cell to reuse amino acids to make more proteins. The proteasome itself is not one protein but many acting together. Proteins frequently interact to form larger structures with important cellular functions. For example, the tail of a human sperm is a structure composed of many types of proteins that work together to form a complex rotary engine that propels the sperm forward.

      Future research about protein folding and misfolding:

      Why is it that some misfolded proteins are able to evade systems like chaperones and the proteasome? How can sticky misfolded proteins cause the neurodegenerative diseases listed above? Do some proteins misfold more often than others? These questions are at the forefront of current research seeking to understand basic protein biology and the diseases that result when protein folding goes awry.

      The wide world of proteins, with its great assortment of shapes, bestows cells with capabilities that allow for life to exist and allow for its diversity (e.g., the differences between eye, skin, lung or heart cells, and the differences between species). Perhaps for this reason, the word “protein” is from the Greek word “protas,” meaning “of primary importance.”

      –Contributed by Kerry Geiler, a 4th year Ph.D student in the Harvard Department of Organismic and Evolutionary Biology


      Watch the video: Chapter 19 Su2016 P1 Post-translational processing (June 2022).


Comments:

  1. Makoto

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  2. Daik

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  3. Raghib

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  4. Izreal

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