#50 Biochemistry Gene Expression II Lecture for Kevin Ahern's BB 451/551

Uploaded by oharow on 10.03.2012

Kevin Ahern:... have class in a building like this
when it's so nice outside?
No, I'm not either.
Alright, so a couple things.
One, I keep forgetting to bring notecards.
You will get notecards for the exam.
Like last term you'll need to get them from me.
And if somebody is really nice and reminds me to bring
them on Monday I will bring them on Monday
so you can have them to work on.
Somebody reminded me actually on Wednesday
but I didn't bring them today.
Anyway, so notecards.
And as I said last term, you have to get them from me
and you have to turn one in whether you use it or not.
So make sure you get a notecard from me.
We're moving our way through gene expression
and we talk about a couple things
in prokaryotic gene expression.
I'm going to finish those up and then say a few
things about eukaryotic gene expression and get you
hopefully out of here in a reasonable fashion today.
So we'll try not to do too many things.
When I finished last time I was at about this point
and I showed you what happens with bacterial cells
when they encounter lactose.
And the point of this slide was to simply show you
a phenomenon known as induction.
When I was your age induction meant something very different
than it means with what you see on the screen.
Induction meant you were getting drafted
to go to a war in Vietnam, and so this is a much
better kind of induction.
Induction happens when some cells receive
some signal of some sort that make them do something.
In this case what's happening is this enzyme
to break down lactose is being induced.
It's being synthesized where it wasn't being
Synthesized before and the signal turned out
to be coming from lactose itself.
So when lactose appeared in this cell's food
then the enzyme to break it down was synthesized.
How does it work?
Well there's really only one enzyme the cell
needs to break down lactose.
It turns out that there's an operon that has three genes,
and I'll say a little bit about those in just a bit,
but there's only one of those three
that's needed to break down lactose.
And it's an enzyme called beta-galactosidase
as you can see right here.
Lactose, you recall, is a disaccharide.
It has two sugar residues in it,
one glucose and one galactose.
And so in order to metabolize lactose
cells have to break it down.
And then you remember glucose, of course,
can go through glycolysis and galactose
can get converted into intermediates in glycolysis
so that it can get broken down as well,
but this has to happen.
So cells don't want to make beta-galactosidase
if there's no lactose because that's a waste of energy
and they want to make a lot of beta-galactosidase
when there is lactose present.
So we have to understand how it is that they do that.
Before I tell you about how they do it
I want to tell you something about a very cool trick
that biochemists came up with.
I mentioned last term that biochemists
are basically lazy people, and they like to find
the easiest ways that they can to measure something,
and we talked about this with respect to serine proteases
where we saw a serine protease could cleave
an artificial substrate that gave a yellow color
and so we could see how much of the enzyme was actually
working by measuring how much yellow color was produced.
Well similar things have been created for studying
beta-galactosidase, and what you see on the screen
is a molecule that we call X-Gal and it looks enough
like lactose to fool beta-galactosidase.
So beta-galactosidase, when it encounters X-Gal,
will treat it as if it were X-Gal and it will cleave
the bond between this guy right here
and this red residue on the side.
Well the upshot of that is if this cleavage occurs
a molecule with a blue color will be produced.
So like we did with the serine proteases
we can measure the amount of beta-galactosidase present
by simply measuring how much blue color is present.
And that turns out to be a very,
very useful tool as we shall see.
So beta-galactosidase can be measured very easily in that way.
I said a little bit about operons and now I want
to show you up close and personal
the beta-galactoside operon.
So we're looking at a segment here of the E. coli chromosome,
and in this segment of the E. coli chromosome
we have several things that are important.
I want to emphasize that the operon itself
is located from here to the right,
that is from the purple-actually from here,
the purple to the right.
This is a blow up of this region here.
From this guy to the right or in essence
from this to the right.
So the operon itself is a coding region.
It codes for three different genes.
Why do I show you this guy over here, this purple region?
This purple region on the left is not part of the operon
but it codes for a gene that helps to regulate that operon.
What's the difference between being
over here and being over here?
If you recall my definition of an operon
I said that an operon is a collection of genes
that are all controlled by a single promoter.
The promoter is right here and so there's no way
that this promoter can make that gene.
So this gene over here called I is not a part of the operon.
But I encodes something very important.
In fact what it encodes is the lac repressor.
So the lac repressor is, its coding region
is right next to the lac operon.
How convenient, right?
And the operon is over here.
Well for our purposes, we're very interested
in this gene right here, lacZ because Z is the gene
that codes for the beta-galactoside.
There are two other genes that are present
and for our purposes they don't play a very important
role but I'll tell you what they are.
One is a protein called a permease.
The permease allows the cell to more
efficiently absorb lactose.
It allows the cell to more efficiently absorb lactose.
So when the cell detects that there's lactose
there it makes permease which lets even more lactose
in and it makes beta-galactoside so that it can break it down.
Last, the cell has an odd enzyme called a transacetylase
which you don't even know the name of,
and the transacetylase modifies lactose slightly.
And when it does it creates a byproduct called allolactose.
Now this gets a little confusing and for our purposes
we will assume that this allolactose is the same as lactose.
For our purposes we're going to assume that.
If you're curious about why I make that assumption
or are curious about how this actually occurs
I would be happy to tell you separately,
but to keep things simple we're going to treat
allolactose as if it's lactose.
So these are the three genes of the lac operon.
You'll notice that there are control sites
and the control sites are actually enlarged
a little bit so we can see down here.
We see P and O and your book is using that to stand
for "promoter" and "operator" although I tend to say
that the operator is really part of the promoter.
So promoter and operator.
The operator is that region that you think
about where the lac repressor binds.
How does this operon get regulated?
Well it turns out to be fairly simple.
Student: Is the gene Y, is that where the permease comes from?
Kevin Ahern: Yes.
How does this operon get regulated?
Well it starts out with a negative regulation
and negative regulation means that lac repressor
when it's present in the cell, and there's no lactose present,
will bind to the operator.
The lac repressor binds to the operator,
RNA polymerase cannot bind, and as I said last time,
transcription is completely turned off.
There's no synthesis of this operon.
However, when the cell encounters some lactose,
lactose in a modified form gets bound to the repressor.
The repressor cannot only bind to the operator,
it can also bind to a modified form of lactose
and we're going to treat it again
as if it's just simply lactose.
What happens?
Well it can't bind to both.
When the repressor binds to lactose it lets go of the operator.
And this is a signal to the cell that the promoter's wide open.
Now we can start transcribing the operon and the cells
only transcribe the operon when lactose is present.
If lactose is absent the repressor turns off.
If lactose is present the repressor is gone
and RNA polymerase jumps in and starts making the operon.
It's a very simple but very, very useful system.
One molecule, bang.
The cell detects it's there,
"Oh, it's time to start making enzyme."
Very very cool.
Very simple.
There's the lac repressor bound to DNA.
This is actually the allolactose I talked about.
This is actually the molecule that binds to the repressor.
We're not going to focus on it,
we'll treat it as if it's lactose.
So don't sweat how do you get lactose
if you don't have the operon going, alright?
Last I need to tell you about this guy here.
So let's say I'm a researcher and I want to study
the way in which the lac operon works.
I want to study the way in which the operon works
and I've got a bit of a problem.
I give cells lactose, what's going to happen?
Well those cells, lactose is going to bind to the repressor,
it's going to start synthesizing the operon,
and what's going to happen to the concentration
of lactose over time?
It's going to fall.
So I've got two variables in my equation.
I want to study this operon so that I see
what happens when lactose is present,
but then lactose itself starts disappearing.
I've got a problem.
It would be nice to be able to find something
that binds to the repressor that doesn't get broken down.
So I can see what happens with this over time.
And so that's what this molecule is.
It's called IPTG, isopropylthiogalactoside.
I never can remember that myself so you'll probably
call it IPTG like I do.
IPTG has the very useful property that it will bind
to the lac repressor just as if it's lactose.
But, importantly, beta-galactoside won't break this down.
So once I get this to induce the operon
the operon just stays on.
There's no running out of the lactose material
that would be needed to bind to the lac repressor.
So now I've got an inducing molecule.
It's artificial, it's manmade,
it's not a natural product of any sort.
It's a manmade thing but now I've got something that I can
turn on the operon and it won't itself get broken down.
That's really useful.
So two things I've given you are artificial, X-Gal and IPTG.
IPTG is an artificial inducer,
X-Gal is an artificial substrate.
X-Gal gives me a blue color,
this guy's going to give me beta-galactoside.
Are we clear?
Now this turns out to be really useful
for biotechnology purposes.
Let's say, for example, that I have a gene
for a human growth hormone and I want to put it
into bacterial cells and I want the bacterial cells
to make a lot of human growth hormone and I want to control it.
I'm a control freak, right?
So what do I do?
I take the lac promoter, I throw away all the operon,
and I put my gene in front of the lac promoter
and the lac operator.
I let these cells grow up and what do I do?
I treat them with IPTG and what's going to happen?
They're going to start transcribing my gene
like crazy and they can't break down the IPTG.
They're going to make a lot of messenger RNA for my gene.
They're going to start synthesizing my gene
and make me a very cool product.
So this turns out to be very useful for biotechnology purposes.
Student: So will IPTG bind more than just the lac operon?
Kevin Ahern: Will IPTG bind more than the lac repressor
I think is what you're asking, and in general, no.
Student: So do you have to have a different
one for human growth hormone?
Kevin Ahern: Well all that you care about is that
your making the messenger RNA for human growth hormone.
So if you put the human growth hormone in front
of the promotor then this is turning on the promoter,
you're making messenger RNA for human growth hormone.
That's going to be translated into human growth hormone
and the aim here is you're making a bunch of RNA.
Make sense?
Yes sir?
Student: Wouldn't you have to at the same time engineer
some export properties into the cell or lyse
them all at once to harvest your product?
Kevin Ahern: Yeah, so there's other considerations.
Would you have to export it from the cell?
Would you have to bust the cells open?
There's a lot of other considerations in terms
of getting your final product.
But this is a way to make your final product
that's going to be useful to you, yeah.
Well let's see.
This is simply reminding us that when,
here's the lac repressor shown in purple,
when there's no lactose present the repressor
binds to the operator, the operon's turned off.
When lactose is present this inducer molecule binds to it.
This repressor can no longer bind to the operator.
The RNA polymerase comes in and does its thing,
make a lot of beta-galactoside,
permease, and the transacetylase.
Interestingly, if we look across the entire E. coli genome
of six million base pairs, that's million, not billion,
six million base pairs, and we look for are there
other things that use the lac promoter,
for example things that might be turned on
when lactose is present, we see that there's nothing else.
The only place we see the lac promoter
in the E. coli chromosome is where the lac operon is.
We don't see it for other things.
It's only there.
And that's not true for all promoters.
Here's a promoter that is necessary for making purines.
That's why it's called a p-u-r operator.
We see that promoter appearing in a variety of places.
Why would we see one promoter appearing
in a variety of places and another promoter
only appearing in one place?
The answer is the need to coordinate things.
So if I'm going to make purines it might take
a bunch of different genes in different places
on the chromosome to be made.
So let's imagine I've, you saw when we went through
the nucleotide metabolism you saw it took about ten genes.
It took a lot of things to make purines.
We might imagine that, well here's a few of them over here,
here's a few of them over here, here's a few of them over here.
Once I have the signal that says,
"Hey, it's time to make purines," all of these different
operons get turned on.
Whereas in the case of lactose metabolism,
the only thing we really need for lactose
metabolism is beta-galactoside.
We don't need anything else.
It works on one operon and we've got it.
Well there's yet another piece to this puzzle
of the lac operon and it's interesting.
It's interesting.
The promoter of the lac operon is not,
underline not, an ideal promoter.
We talked about the TATA box and how the closer
the sequence was to that TATAAT the stronger the promoter was.
Well if you look at the lac promoter it's not very strong.
It doesn't look very much like TATAAT.
So if we rely simply on that promoter sequence
and nothing else we would get some synthesis
of messenger RNA but not an awful lot.
Well it turns out that there's something else
in the lac promoter region that really turns
on the transcription, and what it is, is a binding site
for a protein called CAP, C-A-P.
CAP is also in some places called CRP.
And basically what it is, is a protein that when
it binds to cyclic AMP binds to this region
next to the lac promoter.
You see it depicted here and what it does is these CAP
or CRP proteins favor the binding of RNA polymerase.
They act like a super sigma factor.
They really help recruit and get RNA polymerase
to come in there and start transcribing messenger RNA.
So because of CAP the lac promoter turns out to be very strong.
Does CAP have, does cyclic AMP play a role in this process?
It does.
Is cyclic AMP present when cells have low energy?
And the answer is to some extent, yes it is.
Now the next question is if I have CAP binding
and I have a lac repressor binding, who wins?
It turns out they can both bind and the answer
is the lac repressor wins because as much
as this would try to load it on,
if the repressor is sitting right here
there's no place for the polymerase.
So most of the time the cell,
or I shouldn't say most of the time,
but a good deal of the time the cell actually has CAP
protein sitting there waiting for things to happen.
But if you think about it, it makes sense.
The cell doesn't want to transcribe this operon
unless lactose is present and the repressor
won't leave until lactose is present.
Very simple, very cool system.
Now I'll stop there and take questions
since there's usually questions about that.
Yes sir?
Student: So CAP binds to the promoter?
Kevin Ahern: CAP binds to the region of the promoter,
that is correct.
Pretty cool.
Orienting yourself, CAP is up here,
RNA polymerase is here, and the lac repressor
binding site's about right here.
That's the physical orientation of them on the chromosome.
And of course the gene's going to be down here.
The operon's going to be down here.
Good, cool.
That's the binding.
Not surprisingly CAP binding induces a bend in the DNA
and you've seen the role of bends in DNA
in terms of opening up strands.
That's going to be important for RNA polymerase
and it's one of the ways that CAP binding
facilitates the binding of RNA polymerase.
So that's what I want to say about the lac operon.
I'll actually come back and say a little bit
about it later with respect to eukaryotes,
interestingly enough more as a tool
than as a phenomenon in eukaryotes.
Now I want to spend just a minute,
and I'm not going to spend much time on this
because I think it's to be honest with you a bit
overrated but I want you to have an idea
of other types of regulation that bacterial cells have.
Bacterial cells' regulation is relatively simple
and this one you'll see is, "Oh, it's not very simple,"
but it actually is fairly simple in terms
of structure and requirements.
But I want to show you something because it tells
you something very cool about amino acids in cells.
What I'm showing you on the screen is a depiction
of the 5' end of the messenger RNA of the tryptophan operon.
So the tryptophan operon is a very long
sequence of RNA that gets made.
It's about ten different genes that are in this operon
so it's a pretty long messenger RNA.
And the operon is involved in the proteins,
I should say the proteins that are coded by this operon
are involved in helping the cell to make tryptophan.
So the cells have to make a decision.
Do I need to make tryptophan or not make tryptophan
because I don't want to be making this very long
messenger RNA unless I really need tryptophan
because I'm again I'm going to be wasting
energy if I'm not careful.
Well the way that the cells make this decision
is by this phenomenon I'm getting ready
to describe to you called attenuation.
Now your book doesn't have the best figures
for this and it shows you two possible structures
that can exist in this region.
So let me show you what we're looking at here.
We have...I said there's ten different genes
that are in this messenger RNA.
Very close to the 5' end there's a little tiny,
look how short that polypeptide is,
there's a little tiny coding region for a polypeptide.
And two of the amino acids in there specify tryptophan.
These two amino acids the cells use as a barometer to see,
to determine how much tryptophan's present in the cell.
You also see two regions further downstream,
one that's shown in red and one that's shown in blue.
These are called the region of the attenuator.
Now what I don't like about what your book does
is it doesn't show the most important thing
which is that a portion of the red over here on the right
can actually interact with a portion of this blue.
So we really have three possible structures.
This one, or this one, or a sort of a hybrid between the two.
That hybrid between the two turns out to be important.
How does this work?
Well let's imagine that the cell is in a situation
where it has plenty of tryptophan.
It doesn't want to be making this operon
but the control of this expression of these genes
is not at the level of transcription,
at least not at the level of initiation of transcription.
Rather it's at the level of termination of transcription.
So the question the cell has is-
it's always going to initiate it.
The question is is it going to terminate it early
or is it going to terminate it
only after all the genes have been copied?
It's going to want to terminate it early
if there's plenty of tryptophan
and it's not going to want to terminate it
until all the genes have been copied
unless there's low tryptophan.
Well what you see on the screen is a depiction
of what happens when there is plenty of tryptophan.
What happens?
Well RNA polymerase starts making the messenger RNA.
And it's gone and it's made all this stuff up here.
And you recall that we're in prokaryotes
so once we've started getting to coding regions
the ribosome can come along and start translating behind there.
So the ribosome is following the RNA polymerase along,
doing its thing.
And what's happened is if there's plenty of tryptophan
the ribosome just goes scooting through this little region.
Remember there's two tryptophans in this little peptide.
It doesn't get slowed down waiting for tryptophan
because there's plenty of tryptophan
so the ribosome comes scooting through.
And when the ribosome comes scooting through
it encounters this red region and the red region
when it's covered up with the ribosome
can't do that sort of red/blue pairing that I talked about,
and instead the blue pairs with itself.
Well the blue, guess what, is a terminator.
We talked about those factor independent sequences
that can lift the butt end of the polymerase up
and kick the polymerase off and everything falls apart.
Look what happens.
When there's plenty of tryptophan termination happens early
and we only make this one little tiny sequence.
We don't make all the others.
The cell doesn't waste too much energy.
Now let's imagine what happens
when there's very little tryptophan in the cell.
When there's little tryptophan
the ribosome starts doing its thing and it gets to that region
where there's two tryptophans
and it says, "Uh, where's the tryptophan?
"Where's the tryptophan?
"Where's the tryptophan?"
It's sitting there waiting.
The tryptophan concentration's low
and remember we're talking about a diffusion process
to get those tRNAs in there, so if tryptophan's low
the ribosome's going to sit here and wait for awhile
for the transfer RNA with the tryptophan on it
to diffuse into the A site.
Because the ribosome is slowed, what happens?
Well what happens is basically, and this is a little inaccurate,
what basically happens is this red guy
pairs with this blue guy
and stops the blue guy from being a terminator.
In the first case we had covered the red guy up
with the ribosome.
In this case the red guy's not covered up.
So we can think of the red guy as an anti-terminator.
When the red guy forms like this
or the red guy forms the red/blue complex
there's no termination.
Now RNA polymerase says, "Okay,"
and now it goes merrily along its way
and makes the entire operon.
So what I've just told you is that when there's low tryptophan
there's no termination, when there's abundant tryptophan
there's early termination.
I'll stop there and take questions.
You guys are really quiet today.
Yes, Karen?
Student: I don't really understand the process
behind how that terminator blue region forms.
Kevin Ahern: Yep, good question.
So she doesn't understand when does it make a blue duplex
and when does it not make a blue duplex?
The answer to that question
is based on the position of the ribosome.
And that's why the red sequence is important.
That's why I don't like this figure.
Let's go back to the previous figure.
The previous figure, the ribosome is moving merrily along
because there's abundant tryptophan.
It doesn't get stuck over here.
It goes and it covers up part of the red sequence,
the anti-terminator.
The anti-terminator can't stop the blue from forming.
As a consequence of that the blue forms, termination happens.
On the other hand, when the ribosome gets stopped right here
there's nothing to stop this anti-terminator
from anti-terminating, as it were.
As I said, in reality the red is pairing with part of the blue
and when the red is pairing with part of the blue
the blue can't pair with itself.
That means then that no termination happens
when there's low tryptophan.
Does that help?
Student: A little.
Kevin Ahern: A little.
[Kevin laughing]
Student: It's more like the mechanism behind
what causes it to hold it on itself.
Are they just like complementary sequences to each other?
Kevin Ahern: Very good question.
So what cause it to fold in on itself?
If the pairs, if the bases there can, they will.
The sequence is always the same so the question is,
is the blue free to itself only
or is it having to interact with this red guy?
And so the ribosome really determines
whether or not the red guy is available
to block that blue from pairing with itself.
Does that make sense?
Other questions.
Student: So that little chunk out front,
was that just the little short ten- or fifteen-chunk we saw
or is there like one or two genes
that it does get through copying?
Kevin Ahern: No, his question is are there other things up there
besides that ten- or fifteen-amino acid sequence.
That's it.
So there's ten or fifteen, there's a little space,
then there's another gene, another gene, another gene,
a whole bunch down the stream.
So we're looking at a short region of noncoding
between this first little segment and the next gene.
It's a very cool system.
Now it's not just an oddball system.
It turns out that several amino acid operons in E. coli
are regulated in exactly the same way.
This shows the operon region for several.
You can probably look at this
and tell me which operon is involved in A.
I hope.
Especially since it's in red up there.
Look at the threonines.
Why does the cell have all those threonines there?
Well if threonine's in low concentration
the ribosome's going to slow down going through here,
it's going to let that red region interact with the blue
and no termination will happen, the operon will obey.
So as a consequence when threonine is low
the cell's going to make the threonine operon.
When phenylalanine is low
the cell's going to make the phenylalanine operon.
When histidine is low
the cell's going to make the histidine operon.
Simple system.
Very very efficient at helping the cell
to make the right amount of a given protein.
Yes sir?
Student: Is there any particular reason why it's all amino acids
that have relatively large sidechains?
Kevin Ahern: No, there are some.
Valine for example is also in this category.
So it's not just coincidence
that these have large sidechains, no.
Let's see.
You guys want to sing a song?
Students: Yes.
Kevin Ahern: So I actually have two songs today.
If we get far enough we'll do a second one.
The first one is-let's see, this is the first one here.
The first one some of you may know.
It might not be a big one.
The song Maria from West Side Story?
Students: Yes.
Kevin Ahern: So this one's a little theatrical
so we'll have to get silly.
Lyrics: Translation!
The most intricate thing I ever saw
Kevin Ahern: Here's where you come in.
Lyrics: From five prime to three prime, translation, translation
The final step that we know about the central dogma
Amino, carboxyl, translation, translation
Translation, translation, translation!
I just learned the steps of translation
And all the things they say
About tRNA are true
To form peptide bonds in translation
The ribosomal cleft
Must bind to an E-F-T-u
AUG binds the f-met's cargo
16S lines up Shine and Dalgarno
I'll never stop needing translation
The most intricate thing I ever saw
Kevin Ahern: Thank you.
What's that?
I always start too high, that's my problem.
[class laughing]
If we get far enough we'll have another one
before the end of the period.
Let's talk about gene expression in eukaryotic cells.
And in eukaryotic cells I hope by the time I'm done with this,
and I'm not going to finish this today,
but I hope that by the time I'm done with this
you'll get a feeling for the complexity
of what eukaryotes have to do in regulating gene expression.
Eukaryotic gene expression is really governed by two
very, very important things.
One is differentiation.
Different cells in an organism
have very different protein needs.
The other is that the DNA in eukaryotic cells
is not bare naked like it is in prokaryotic cells
but it's wrapped up with histones
and it's very, very complicated structure.
Getting the factors into the DNA to facilitate transcription
is a much bigger problem than it is in prokaryotic cells.
Well let's first of all
talk about those proteins that are there.
If we take eukaryotic DNA
and we start peeling apart the chromosomes
very, very carefully what we discover
is they look something like this in an electron micrograph.
What you see is a structure
that people describe as beads on a string.
And what you're actually seeing is DNA strand
wrapped in a coil around cores of proteins.
Core of protein, core of protein,
core of protein, core of protein.
And these cores of proteins contain proteins called histones.
Histones are basic charge, that is they're positively charged
at physiological pH.
And DNA is negatively charged at physiological pH.
They have a very strong attraction for each other.
And each of these sort of central wrapping things here
has a total of eight proteins,
two copies each of four different proteins.
The four proteins in there are known as H2A, H2B, H3, and H4.
And as I will show you in a little bit
the structure of those proteins
are very very similar to each other.
Now there's the beads.
The string has DNA but the string in some cases
has another histone on it.
It doesn't cause the coiling though.
The other histone that we frequently find
in the spacer regions is called H1.
So there are five different histone proteins for our purposes.
Five different histone proteins,
and the region between the beads is frequently occupied
by the histone protein known as H1.
The structure of the wound DNA around the eight proteins
on the center create something
called a nucleosomal core particle.
That's the most fundamental unit of chromatin.
What's chromatin?
Chromatin is simply the DNA-protein complex.
So the most fundamental unit of chromatin
is a nucleosomal core particle.
This region that you see here
is roughly two hundred base pairs in length
and you can see that the DNA wraps around it a couple times
and that's what you're seeing in those beads on that string.
There's a sideview.
And that's a schematic.
DNA wrapping, wrapping here.
So we see it's making some wraps
around those individual proteins that are in there.
Now this is what those proteins look like.
We talk about conservation of structure.
Four different proteins
all involved in that nucleosomal core particle
and look at their structures.
Really, really similar to each other.
Structure's important for function.
The fact that we see this conservation of structure
says this function's pretty darn important.
Not only do we see this conservation of structure
within an organism, we also see it between organisms.
If we were to compare the histone proteins of yeast,
one of the simplest eukaryotes, to the histone proteins
of human beings we'd have a hard time telling them apart.
So these are really critical proteins.
We don't see many changes.
We know that function is absolutely essential
and not surprisingly.
These guys are having to wrap up those DNAs
so they fit inside the cell.
As I've said in class before the total sum
of all the DNA of a cell is seven feet long.
That's got to be wrapped up.
In a human cell it's seven feet long.
It's got to be wrapped up so it fits.
If it doesn't the cell doesn't have a chance.
The cell can't get bigger.
Those individual core particles themselves
get arranged with each other
and so we start seeing superhelices.
We could have a helix of this
and then we could have a helix of the helices and so forth.
So by the time we see a visible structure in the microscope
like in the form of a chromosome
we're talking about a tremendous amount of packing
and ordering of these things together
to give that structure that we're able to see
in a light microscope.
Well not surprisingly, if the gene that the cell needs to make
is found right here, getting proteins into this
if this is in the middle of a chromosome is not a trivial thing.
The cell has to be able to get proteins in there
to start transcription or proteins in there to replicate DNA
or whatever and so there's a lot of orchestration
that has to happen in order to unwrap this structure
so that transcription can occur.
Now a lot of what I'm going to be telling you today,
or not today but today and on Monday
has to do with the considerations necessary
for unwrapping this structure.
Transcription can only happen
when this structure has been unwrapped.
Well I talked the other day about some DNA binding proteins
and I said that we saw some common features among those.
And the ones I described the other day, the helix-turn-helix,
are mostly found in prokaryotic cells.
There are some other things that we see in DNA binding proteins
and these tend to be found more in eukaryotic cells
and one of which you see on the screen.
So these are protein domains, part of a protein
that is binding to a DNA.
The DNA's at the bottom.
The part of the protein is there.
And look at that.
There's a beautiful helix
that is interacting with the double helix.
You see it says it's a basic region
meaning it's fairly positive and so it's attracted
to and it's binding to this DNA molecule.
This structure called a leucine zipper is really interesting.
It's really interesting because when it was discovered
people were very puzzled by it.
The puzzle was when they analyzed the DNA sequence
about every seven bases they saw a leucine residue.
Leucine, seven bases later, leucine, seven bases later,
leucine, seven bases later, leucine-or not seven bases,
seven amino acids, leucine, seven amino acids, leucine.
Leucine was appearing in a very regular thing
and it was kind of odd.
Why was leucine doing this?
Well leucine you may recall is a fairly hydrophobic amino acid.
It doesn't like water.
It likes to associate with other hydrophobic acids.
And if you go every seven the same side it's appearing on.
So here, here, here, here, here, here,
every seven we're seeing a leucine.
Hmm, that's interesting.
Leucine doesn't like water.
What if I have another strand over here
that has a leucine, leucine, leucine, leucine, leucine, leucine?
What's happening?
The leucine's are interacting with each other
very much like the teeth of a zipper do.
So leucine zippers turn out to be a really important structure
in not only structures within a protein
but sometimes between proteins
and we commonly see them in features like this
when we have something that binds to a DNA molecule.
Another common structure that we see of proteins
that bind in eukaryotes to DNA molecules
are called zinc fingers.
And they're called zinc fingers
because a portion of the protein binds the zinc ion.
Oh it's this thing.
I have a backup recorder in case my microphone goes bad
and it's full.
A portion of the protein binds to zinc
and that causes literally a finger of amino acids to stick out.
And that finger sticking out is the portion
that interacts with the DNA double helix.
So zinc finger structures are also important structures.
If I'm determining the structure of a protein
and I don't know what it does and I see a zinc finger I can say,
"Hmm, maybe it's a DNA binding protein."
Structure can give me some clue
to what the protein actually does.
Yes sir?
Student: Are they generally as triplets like that?
Kevin Ahern: Are they generally as...
oh with the orientation here, yes.
In fact that's a good point.
They will commonly have two or three cysteines
and a histidine that's helping to coordinate
and organize the zinc.
Student: I meant over here with the subunits
you have three zinc finger domains all...
Kevin Ahern: Not necessarily that but this, you will.
Now I need to introduce another term.
A mediator.
A mediator is a person that helps settle arguments, right?
A mediator can help make something happen.
Eukaryotic cells have mediators
and mediators turn out to be really useful
because they can bridge the gap between a transcription factor
and a RNA polymerase.
Remember in a eukaryotic cell
that the RNA polymerase isn't necessarily,
in fact it isn't the first thing that binds.
We have the accessory proteins that bind to the control region
and those accessory proteins
ultimately help the RNA polymerase to bind.
Well what a mediator gives is a bridge
between a transcription factor that might bind
a couple hundred base pairs away and an RNA polymerase
that starts two hundred base pairs away to start transcription.
The mediator will provide as I said a bridge
between the transcription factor protein and the RNA polymerase.
It's a fairly simple concept but that's what a mediator does.
I talked briefly when I talked about transcription
with respect to enhancers and I said enhancers were sequences
that we saw in eukaryotic cells that are very different
than things we saw in prokaryotic cells.
They are things that are bound by proteins
that affect transcription and they affect transcription
in a tissue specific manner.
What does that mean?
Well we see on this particular gene some sequences
that are enhancer sequence elements.
Let's imagine that in skin cells
I have a protein that recognizes CAGCTG.
Only skin cells may have that protein,
and if that's the case then in skin cells
that protein's going to bind in this region
and it's going to activate transcription of a gene
that skin cells will need.
If I go to a muscle cell, it doesn't have CAGCTG,
well it's not going to activate there.
But if the muscle cell needs it,
it might have a protein that recognizes TTAAATTTA.
It might have no proteins that recognize any of these.
So if it has no proteins that recognize any of these
this gene won't be made in a target cell,
in a muscle cell for example.
So enhancer sequence elements allow the cell
to control transcription using specific proteins
that are only made in that cell.
Very very useful control system for cells.
Very simple.
Questions about that?
Okay I said we'd do one other song.
Let's do one other song and we'll get out of here.
It's a fun song.
I think you know the tune to this one.
Everybody likes the Flintstones?
Let's do:
Tiny histones
Wrap up eukaryotic DNA
Using lysine sidechains
They arrange a chromatin array
With them, DNAs of seven feet
Fit inside the nucleus so sweet
When you use the histones
You have to deal with condensation
And its ablation
Inside your chromosomes
Kevin Ahern: See you Monday.
[class murmuring]