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.