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Kevin Ahern: Okay folks, let's get started.
So a couple of things.
One, the exams are available.
And two, I did manage to get a, that shut everybody up quickly,
I did manage to get a grade distribution shown.
The numbers are a little hard to see,
but I think you get an idea about where you stand.
These are the sum of the scores of your two exams.
So I was very, very impressed with the average again.
The average was almost identical to the first exam.
And as I probably have told you before,
when I see a high average on an exam,
I usually see it fall on the next exam.
And you're one of the few classes
I've ever had who did not do that.
So that's really remarkable.
I'm very impressed with that.
You guys also had one very high average last term as well.
So either I'm getting softer in my old age
or you're getting better in your young age,
and I like to think the latter is the case.
That was a very nice performance.
Some of you are struggling, and I recognize that.
And if there is anything I can do to help with that,
of course as always, let me know.
But the class as a whole is doing quite well,
and I'm very pleased with this.
So that's where things are.
The exams, as I said, are available for pick up
in the Ag and Life Sciences Building, room 2011.
And there is a key outside of my office as always.
So I want to go further talking about transcription today.
And we've got quite a bit of ground to cover.
And so I want to get into that
without any further delay shall we say.
Last time I finished,
I had just started talking about promoters.
And promoters are special, important sequences
because they define locations where to start transcription.
It is important to note that the promoter
is adjacent to the start site.
So the promoter is always in the negative numbers
as I showed the numbering scheme last time.
And I pointed out that when we look at,
these are all prokaryotic systems here,
but when we look in prokaryotic cells
and we compare the sequences near the transcription start sites
for hundreds of genes, we see what's called a consensus
that arises of this sequence right here, TATAAT.
And I talked about how the closer that any given,
these are called minus ten sequences,
the closer that any given minus ten sequence was to that TATAAT,
the more effective as a promoter it was.
And by effective, I mean the more copies of mRNA
that were made from it.
So we talk about strength of a promoter.
So a strong promoter is something
from which a lot of RNA will be made, and a weak promoter
is one from which only a small amount will be made.
Now as we will see, there are things that can modify or change
how much is made depending upon circumstances,
but in general, what I've just told you is true.
In addition to sequences at approximately -10,
there's also in E. coli another common sequence
that's seen at about -35.
These are all approximate distances and they are there.
I'm not asking you to memorize these sequences.
I certainly think you should know this is an AT rich region,
but other than knowing that there's a -35,
I don't think memorizing the sequence
really tells you much of anything.
Now why do we see specific sequences?
We see specific sequences
because proteins bind to specific sequences in DNA.
So we don't want random binding.
We want to be able to control, and so proteins have evolved
recognizing and binding to specific sequences.
In the case of E. coli though, that system is pretty simple.
It involves an RNA polymerase.
In some cases it may involve one or two other proteins.
In eukaryotic cells, it's much more complex
and there's an entire orchestra of proteins
that may play roles in controlling transcription.
And I'll say more about those later.
One of the ways in which different promoters can be recognized
with different efficiencies is told by the story
that you see on the screen.
It's not described very well there,
so I hope to clarify that for you.
I said that there were several proteins
that made up RNA polymerase in E. coli.
And I said we weren't going to pay much attention
to what the function of most of those proteins were.
One of those we're going to pay some attention
to is called sigma.
It's called the sigma factor.
Now the sigma factor associates with the RNA polymerase
and what it does is it helps RNA polymerase
recognize a promoter.
So sigma is the protein component of RNA polymerase
that helps the polymerase to recognize
those specific sequences.
Now it turns out that E. coli have several sigma factors.
And some sigma factors will recognize
different promoters than others will.
So now we start to see that as things change,
if we change the sigma factors,
the RNA polymerase may start recognizing
and transcribing other genes
compared to when that given sigma factor is not present.
So most of the time the sigma factor
that is there likes the TATA box.
It helps RNA bind to the TATA box, and everybody's happy.
But there are circumstances that cells go through
where they have to respond to environmental change.
And that's something cells have to do a lot of.
The more able they are to respond
to specific environmental changes,
the more likely they are to survive.
One of those environmental changes that happens
is known as heat shock.
I talked a little bit about heat shock last term
when I talked about folding of proteins.
When we think about what happens when we heat up proteins,
what happens, what'd you learn from
last term when we heat up proteins?
Student: They denature.
Kevin Ahern: Yeah, they denature.
So we could imagine that if a cell gets a minor heat shock
and it can't respond to that, it may die.
Certainly if we get it too hot, if we boil everything,
then the cell isn't going to be around to do things.
But if the temperature goes up three or four degrees,
it would be useful for the cell to be able to respond to that
because that's not a drastic change.
In E. coli, it turns out that they have a special sigma factor
that is made in response to heat shock.
So when heat shock occurs, a different sigma factor than normal
is made by the cell.
We're not going to go into the mechanism
about how that's controlled because it's fairly complicated.
But suffice it to say that when a cell gets heat shock,
it makes a different sigma factor.
Why is that significant?
That different sigma factor interacts with RNA polymerase
and now, instead of taking RNA polymerase
to all of the regular TATA boxes,
it takes it to a different set of promoters
and helps them to be transcribed.
You see a couple of them on the screen.
Here's a heat shock promoter.
Look at that.
It doesn't look anything like a TATA box,
but this sigma factor will recognize this.
So let's think about...Yeah?
Female student: What's the Ns?
Kevin Ahern: Ns mean any.
Female student: Any?
Kevin Ahern: Any nucleotides.
Could be G, A, U, or C.
So whenever you see N in a nucleotide sequence,
it means any of the four bases.
Now what does this mean?
It means that if the cell is not experiencing heat shock,
the regular sigma factor will not recognize this,
and it's not making heat shock proteins.
However, when heat shock occurs,
a sigma factor is made that recognizes this,
and now there's a whole set of heat shock
proteins that get made.
And these heat shock proteins
help the cell to survive the shock of heat.
So I talked last term a little bit about chaperonins.
Anybody remember what chaperonins are?
What do they do?
Jodi: Help protein folding.
Kevin Ahern: They help proteins to fold.
Chaperonins, they have heat shock promoters.
So E. coli gets heat shock, it has gotta unfold
some of its proteins, but now what does it do?
It starts making chaperonins to properly fold proteins.
It's a very cool system.
Now heat shock is not something that's unique to E. coli.
Virtually every cell on the face of the Earth
has a set of heat shock proteins
that allow it to respond to minor heat changes
that would otherwise kill it
if it didn't fold its proteins properly.
We have heat shock genes in our cells for example,
which is good because we get fever, right?
So different sigma factors allow different things.
Here's one for nitrogen starvation.
We can imagine that when the cell starts running
out of nitrogen, it's a different set of conditions
than it would otherwise have.
And so when that happens, a sigma factor
that recognizes this promoter to turn on the genes
for making the response to nitrogen becomes available.
Now in E. coli, these systems are very simple.
We say, "Well, there's one sigma factor here
"that's made under this condition.
"There's a different sigma factor that's made over here
"under this condition."
In eukaryotic cells, as I said, there's an orchestra.
There are hundreds of possible iterations
of different proteins and combinations that can be made.
And no, you're not going to memorize them all.
But we will talk about them in general when we get to there.
In E. coli, it's relatively simple.
Last time I went through sort of a verbal description
of what you see on the screen, which is what's happening
during the process of transcription.
During transcription, RNA polymerase has to be copying strands.
And you can see that process sort of happening here.
I think this exaggerates the size a little bit.
But suffice it to say we can see the process occurring.
This is a segment of DNA that is being transcribed,
meaning that RNA is being made, and it's being made
by this big yellow guy here, the RNA polymerase.
You'll see that the DNA strands have been pulled apart
as I've described.
There up here is the coding strand and down here
the strand being copied is the template strand.
You'll notice that the RNA-DNA duplex
does not extend for the entire length of the RNA.
There's this floppy end that's hanging off of here.
So the further that this RNA polymerase moves along,
the more unpaired this guy gets here,
and more floppy end hangs off,
and the more this duplex has to reform.
Now if we think about what's happening here,
ahead of the RNA polymerase, what's happening?
In essence, the strands are getting pulled apart
because we have to move forward.
If we're pulling apart strands, what's going to happen
ahead of that bubble?
We're going to have strain.
We're going to have a lot of twists put into there.
So there is, in fact, a topoisomerase in front of-
this is called the transcription bubble.
We talk about replication fork for DNA.
We talk about transcription bubble for making RNA.
There is a topoisomerase ahead of the transcription bubble
that alleviates that strain.
Look what's happening behind there.
We have to start rewinding everything, remaking the duplex.
We're going to have the opposite phenomenon over here.
Up here we're going to have too many twists,
and back here we're not going to have enough.
And yes, it takes a topoisomerase to fix that one as well.
So on either side of a transcription bubble,
we have topoisomerases that are adjusting the base pairs
per turn appropriate so that the DNA will be relaxed.
This process occurs, when we look at transcription,
and we'll talk about a similar thing with translation,
we'll describe these as happening in three general phases,
initiation, elongation, and termination.
With E. coli, that initiation phase is studied considerably.
And though I don't have a figure to show you about that,
I will tell you a little bit about what happens.
In fact, I've got an interesting protein here.
Not a protein, an antibiotic.
My mind isn't working.
It's called rifampicin.
And rifampicin is a molecule that can bind to E. coli
RNA polymerase, and it stops the E. coli RNA polymerase
from being able to progress
from the initiation to the elongation phase.
There's actually a stepwise process
in which these processes occur.
The initiation phase occurs
during about the first ten nucleotides being synthesized
in E. coli RNA.
The first ten nucleotides or so.
During that time, the cell is setting up
what is called an open complex.
That is the strands have been opened, and that open complex
is being made during that initiation phase.
The first few nucleotides are put down.
Once those first few nucleotides get put down,
the sigma factor goes bye-bye.
So we got the first ten nucleotides down.
Once the sigma factor has gone bye-bye,
we are in the elongation phase.
What rifampicin does is it prevents E. coli
from progressing correctly through that initiation phase.
It never gets to elongation.
And so it stops transcription in its tracks.
Well, if you stop transcription in its tracks,
you are going to, of course, kill cells
and that is why rifampicin is an antibiotic.
Elongation, is kind of like my bumper sticker the other day,
That is basically what happens.
It just goes on until we enter the termination phase.
Now as I told you what happens with the RNA polymerase
in E. coli is it works in fits and starts.
Up, back, up, back, up, back.
And this is continuing throughout the elongation phase.
Moving like this, up, back.
Somebody asked me yesterday, "When it moves backwards,
"does it chew out nucleotides?"
And the answer is yes, it does or yes, it can.
We'll see that there are some considerations
of the elongation phase
as they relate to termination in a minute.
But I want to turn our attention now to the termination phase.
Then I'll come back and relate that to elongation for you.
Termination, of course, is the process
whereby we are stopping transcription,
and termination is every bit as important as initiation.
We don't want to copy the entire
E. coli chromosome in a single RNA.
A waste of energy.
A waste of time.
And besides, we don't want to make all those proteins
at the same time in the same amounts.
So having a specific initiation point
and at least a relatively specific termination point
is in the cell's interest.
It is true whether it is a prokaryotic cell
or a eukaryotic cell.
There are two general mechanisms
I want to talk about for termination.
They're called factor dependent and factor independent.
The factor independent is the first one I will talk about.
And it arises when an interesting structure,
like you see on the screen, appears in the RNA.
Now I'll describe a little bit about this.
Everything you've thought about with DNA base pairs so far
has involved two different strands.
One strand up here base paired to another strand down here.
However, it is entirely possible for base pairs to form
within a given strand if the bases themselves
are complementary to each other.
You can see that this is an RNA
in which what is called a stem loop,
There's a stem, there's a loop,
that is formed because these bases
can actually form base pairs with each other.
Now this stem loop has a specific sequence
in addition to having a specific structure.
And what this guy does is as the E. coli RNA polymerase
is moving along, it's copying the DNA, it's making this RNA,
it's making this RNA, it's making this RNA
and by the time it starts getting back down here,
these bases start seeing each other,
and they start forming a duplex.
I like to think of this thing like the jack of your car.
When you have a flat tire, you get the jack of your car out,
and you jack up the car so that you can take the tire off.
This guy provides lift just like a jack does.
Remember the RNA polymerase is sitting right here.
It's making this thing.
And the DNA strand it's copying is down here.
You got that picture in your heads?
This jack lifts up the butt end of the RNA polymerase.
So where the RNA polymerase was sitting nice and conveniently
on the DNA and making this thing,
now its butt end has been lifted up.
It no longer is sitting properly on the DNA.
That starts the process whereby the RNA polymerase falls off.
It falls off.
And something that helps it to fall off is the fact that,
look at the sequence of bases it just finished up with.
A, U, U, U, U.
The weakest hydrogen bonds.
This is the least stable duplex that we can have.
The RNA itself is ready to fall off.
Everything is ready to fall off,
and you've terminated transcription.
Now this is called factor independent
because there is no protein that is necessary
to make this happen.
No protein is necessary to make this happen.
The sequences themselves are built into the DNA,
which of course is made into the RNA.
And as a consequence, the RNA polymerase falls off.
These fall-off sequences happen at very specific places.
That is we have very specific points of termination
when we have factor independent termination of transcription.
Now some of these can be kind of elaborate and kind of cool.
So one of these that is out there,
I'm going to show you what looks like a complicated figure,
but it's actually pretty straightforward.
This is a gene for making a Flavin, like FAD,
only this one is called FMN, Flavin mononucleotide.
Don't worry about the name.
Here's the RNA for this Flavin.
If we look over here, this is a termination loop.
This stem loop will act like a jack if it gets a chance to form.
If it gets a chance to form, it will terminate transcription.
Everybody understand that?
So the question that we're asking on the screen
is under what conditions will this form, as we see on the left,
or not form, as we see on the right.
Well, on the left, it will form
only when there is abundant FMN.
Because FMN will sit in this complicated RNA-looking structure
and keep these sequences here
from interacting with these sequences over here.
As a consequence, these sequences over here
can interact like that and make the stem loop.
When we have plenty of FMN, what's going to happen?
Termination is going to happen,
and it's going to happen right here at this point.
It's going to happen very early.
The cell is not going to make anymore RNA
for making this protein that makes FMN
because there is plenty of FMN.
On the other hand, if there is a shortage of FMN,
there's nothing to stabilize this structure
and now those sequences that we saw base paired over here
get tied up in this bigger structure.
This guy will not terminate, at least not at this point,
and the transcription of that gene will proceed.
Now the cell will make the protein that's going to make FMN.
This is a beautiful, simple environmental response.
When FMN is high, transcription terminates early,
the protein is not made.
When FMN is low, transcription proceeds,
and the protein for making FMN is made.
Now I'll stop at that point and take questions?
Male student: You say that loop causes the RNA to fall off.
But to me, on the right, with low FMN,
that looks just like a huge loop.
Kevin Ahern: Well, it does,
but it doesn't have the same structure as this guy does.
So one of the things you'll discover in RNA
is there is a lot of different possible things,
look at this guy right here, that can form.
Because, for example,
you can actually form a stable base pair with G.
See that right there?
So there's a lot more pairing possibilities.
It doesn't make this specific structure.
It is the specificity of that structure that determines it.
Very good question.
A very common question.
So what he's asking is, "Well, look at this guy over here.
"It looks like this jack ought to really be big
"and really ought to kick it off of there very easily."
But it's not that.
It's the specificity of this structure
that actually causes that termination to occur.
Female student: Does the FMN act as like an allosteric factor?
Kevin Ahern: That's a good question.
Does FMN act like an allosteric factor?
Well, we think of allostery, of course,
relating to catalysis in proteins.
So technically, it is not an allosteric factor.
But you're right.
It is acting like one because this is acting as a gage
of how much FMN is there.
How much protein should I make?
So I make FMN or not make FMN.
Jarrod: Does the number of pairs, the RNA nucleotides,
slow the translation of protein?
Kevin Ahern: Yeah, so it's a good question also.
So his question is...
this is called secondary structure by the way.
RNAs can have secondary structure
just like proteins can have secondary structure.
And it has some tertiary structure as well.
This structure that's here can, in fact,
affect how well translation goes on.
So I'll save the answer to that question
until I talk about translation.
But yes, those do play roles in that.
Yes, right here?
Student: So when it's not terminated,
does that include the direction for how FMN behaves
so that it can come back and regulate itself?
Kevin Ahern: That's correct.
So this guy has the perfect feedback mechanism.
If I make plenty of protein, I'm going to have plenty of FMN
and it's going to shut down the making of more protein.
I mean it's a beautiful feedback system.
And E. coli cells have a lot of different ones
very much like this where there's a very simple feedback
to turn on or turn off the synthesis of a given protein.
Female student: So RNA copies from 5' to 3'.
So won't it copy all of that stuff before it gets there?
Kevin Ahern: It going to copy all this before it gets to there.
Female student: So what does all of that copying
make the RNA do?
Kevin Ahern: So her question is,
"Well, it goes and makes all this stuff.
"What happens with this?" Basically.
And the answer is it gets degraded.
It doesn't do anything.
Female student: So the actual part it needs to make the protein
is further out here to the right?
Kevin Ahern: The actual part needed to make the protein
is further out here to the right.
Now, you might say, "Well, that's a pretty good waste of energy."
And it is a waste in a sense, but there are nucleotides here.
But making the proteins necessary for this
are thousands of base pairs.
So the cell invests a certain amount of energy
in being able to use this as a barometer,
which actually makes some sense to do.
Does that answer your question?
Luke: Are there other molecules that will do the same thing,
such as when FMN is high?
Or is it only specific for FMN?
Kevin Ahern: So this particular one is only specific for FMN.
There are similar systems that E. coli uses
to regulate amino acid synthesis.
And it uses a very similar mechanism to this.
Jodi: So is this a second layer of transcriptional control?
I mean does it control initiation of transcription as well?
Or is that just constitutive and this is the only one?
Kevin Ahern: So his question is,
are there other levels of control of transcription
in this particular system?
The answer is no.
And we'll see this happening frequently,
that when cells have ways, needs to control certain genes,
sometimes, depending on the gene,
they may have a couple of different mechanisms in place.
This one only has one, and this is controlling whether or not
that initial sequence terminates or not.
When we look at gene expression,
which we'll talk about later in the term, meaning next week.
When we talk about gene expression,
we discover that not only do we control initiation
of transcription or termination of transcription,
we also control splicing-
which we'll talk about in just a bit.
We control whether protein synthesis starts efficiently.
we control whether or not a protein is degraded.
So there's a lot of different ways that the cell can modulate
how much of a given protein is there.
And the number of different ways tell us, or remind us-
you already know this-remind us
that the level of any given protein is very critical
for a cell to maintain.
Very, very critical.
We don't want to have too much
or too little of any given protein.
Now I've told you about the termination independent.
Let's talk about the termination dependent
because it's kind of a cool one.
The termination dependent system is not quite as precise.
Not the termination dependent.
The factor dependent termination.
The factor dependent mechanism of termination
is not as precise as the factor independent.
You'll see why that's the case in a second.
The factor dependent involves a factor.
And the factor is a protein called rho.
That little sign there is rho.
And rho is a hexameric protein that looks like this.
And you can see it's attached to this floppy tail
that's hanging off of this transcription bubble.
The way rho works is it grabs ahold of the RNA,
and it starts shinnying up it.
It literally shinnies up it.
I always like to give the example of how many times
in gym class and high school did you have to climb the rope.
The first time I did it, I hated that thing.
It was awful, right?
Imagine climbing that rope,
and instead of having the rope attached to something at the top,
somebody is letting it out as you're trying to climb it.
It's a race between how fast you can climb
and how fast someone is letting the rope out.
That's what's happening here.
This RNA is getting longer, and longer.
And rho is shinnying up it.
When rho gets up here, when rho catches up,
and it will catch up eventually, what does it do?
It jacks the butt end up of the RNA polymerase
and everything falls apart.
You've heard that story before.
The factor catches up and allows that to happen.
Well, how does it catch up?
Well obviously, rho is going to have to work faster
than transcription in order for that to happen.
And it does.
And sometimes it doesn't.
What does that mean?
Well, think about how that RNA polymerase is moving,
and slowing down, and moving, and slowing down.
When it slows down,
rho is getting a little bit of a chance to catch up.
And I said there was another factor
about how fast transcription occurs
that I talked about last time.
What's that other consideration that we have?
Kevin Ahern: What's that?
Female student: Types of base pairs.
Kevin Ahern: Types of what?
Female student: The base pairs.
Kevin Ahern: The base pairs.
So what happens when the RNA polymerase hits a stretch of GCs?
It's going to slow down.
When we look at rho dependent transcription termination,
we find that it more commonly occurs in regions
that are GC rich because this is where the rho has had a chance
to catch up with the polymerase and kick it off.
Now rho termination sites are not precise.
They're usually clustered.
So there are several possible places where they can end.
And so we can get several possible RNAs that are made.
But it's a numbers game.
It's not going to make it too far.
Once you go through a few of these,
the likelihood that rho is going to catch up is pretty good.
So factor dependent, factor independent
allow the cell to control where transcription stops.
Jodi: So you have multiple possible endings for this
because it's just sort of a general region
where it's likely to end.
Is that cleaned up later by different splicing?
Kevin Ahern: So his question is
because you have different possible termination sites,
does the cell need to clean that up later?
And the answer is it doesn't.
So it has got no problem with the stuff
that is hanging off there.
Plus this is happening in prokaryotic cells,
and splicing occurs in eukaryotic cells.
Male student: Is there an advantage to factor dependent
versus factor independent?
Kevin Ahern: Yeah, that's a good question.
Is there an advantage to one versus the other,
and why do they have both?
And I don't know the answer to that question.
The advantage I would see is that the factor independent, A,
doesn't require protein and, B, is fairly precise.
But if it malfunctions, so if that stem loop
for whatever reason doesn't jack off the-
bad choice of words - doesn't...
Kevin Ahern: Oh boy.
...doesn't jack up the RNA polymerase, what's going to happen?
Then the transcription is going to continue
and make a much longer transcript.
So it's probably six in one, half a dozen of the other.
I'll make YouTube now, probably, I suppose.
Any other questions?
So much for termination.
We've terminated termination.
What was that?
This shows again where you have various sites,
where you can have various links that are made.
And the more of those sites that it encounters,
the more likely termination is going to occur.
Well, I want to jump down now and talk about tRNA and RNA
in prokaryotes because we're nearing the end of transcription
In prokaryotes there is an important thing to keep in mind.
There's one RNA polymerase.
There's hundreds of copies of it,
but there's only one type of RNA polymerase.
In eukaryotic cells there are three types.
So all of the RNAs that get made in a prokaryotic cell
have to be made by the same RNA polymerase.
These include the ribosomal RNAs, the messenger RNAs,
and the tRNAs.
If you don't know what those are, you should.
So I won't go through those.
The messenger RNAs are the ones we commonly think about
but of course, ribosomal RNAs and transfer RNAs
are absolutely essential for cellular life.
When we look at how the ribosomal RNAs
and tRNAs are made in E. coli, they're generally made
with one long transcript.
And then, the individual pieces get chopped out.
There's a processing that occurs.
So a very important component of transcription is processing.
We'll see in eukaryotes,
there's a lot of processing that goes on.
In prokaryotes, there's not a lot of processing.
But this is one place where processing occurs
because in this system we have to chop out,
we have to cut up specifically the things
that the cell is going to need.
And this happens for ribosomal RNAs and tRNAs specifically.
Now there are some enzymes that help this to occur.
And unfortunately, your book doesn't give us
any figures for that, so I'll just briefly list them.
One is called ribonuclease P.
And it's responsible for chewing away
and making a perfect 5' end of the tRNAs.
Another one is ribonuclease III, and it cuts out the 5S,
the 16S, and the 23S ribosomal RNAs.
Those are three different ribosomal RNAs
that I'll talk about later.
In addition, at the 3' end of tRNAs,
there's a sequence that's added to all of them.
This is a modification that occurs as well.
And there is an enzyme that does that.
I don't have the name of it here.
But suffice it to say that tRNAs need to gain this CCA sequence
at the 3' end.
And the last thing that occurs for our purposes here
is modification of bases.
One of these is uridylate.
You can see some of these modifications here.
Here's a uridylate that has been modified
to be a ribothymidylate.
For all those who tell me, and I get the email from people
who watch my youtube videos,
and they say, "There is no such thing.
"You shouldn't write dTTP
"because there is no such thing as ribothymidine."
Well, there is.
There it is.
And it's not just a fluke thing.
In fact, when we look at transfer RNAs, especially in E. coli,
they get chemically modified to make this.
They get chemically modified to make this.
Transfer RNAs in both prokaryotic and in eukaryotic cells
are significantly chemically modified.
You see uridylate here.
Virtually every base can be modified in a tRNA.
Now it's not completely understood in every case
why some of those modifications are occurring.
It may be that it helps to flag these guys
as being separate from messenger RNAs for example.
It may be that it facilitates or prevents base pairing
in some cases.
But suffice it to say that transfer RNAs
have a lot of chemical modifications that occur to them.
Ribosomal RNAs in E. coli will have some modification as well.
Of the three types of RNA that we have in E. coli,
the messenger RNAs are the least modified.
Very, very little.
That contrasts with eukaryotic cells where there's a lot,
and we'll talk a lot about those.
So in E. coli, it's fairly simple.
tRNAs get chemically modified,
ribosomal RNAs get a little chemically modified,
and messenger RNAs don't get very chemically modified.
No, you don't have to draw these structures or anything.
That concludes what I want to say about prokaryotes.
I'd like to turn our attention now
to say a few things about eukaryotes.
Eukaryotic cells have a very different setup
than prokaryotic cells.
The playing field is very different
for eukaryotic cells compared to prokaryotic cells.
The playing field in a prokaryotic cell,
remember there are no organelles,
so everything is in this cytoplasmic soup that's over here.
What you see happening here in prokaryotic cells
is you see-there's your DNA molecule.
There's transcription occurring.
And what you see down here
is actually you see proteins being made.
This is translation that's occurring down here.
That's a ribosome, and these are proteins that are being made.
We see that even while transcription is occurring
in prokaryotes, translation is also occurring.
And that's because they are both in the same place
at the same time.
That's impossible to happen in eukaryotic cells.
In eukaryotic cells we have a nucleus.
And that nucleus is where the transcription occurs.
And after transcription occurs,
there's a significant amount of processing
that occurs that I'll talk about.
And then, if this is a messenger RNA, it is moved out.
And what happens?
We see translation occurring.
Translation is occurring in the cytoplasm in eukaryotes,
but transcription is occurring in the nucleus.
They're physically separated.
These have some pretty big implications actually
for control of expression.
We'll talk a little bit about those again
when I talk about gene expression.
Eukaryotes, as I noted earlier, have three RNA polymerases.
RNA polymerase I, RNA polymerase II, and RNA polymerase III.
You can see that RNA polymerase I
primarily makes the ribosomal RNAs.
It at least makes all the big ribosomal RNAs.
RNA polymerase II makes the messenger RNAs.
It also makes some of the small nuclear RNAs
that I'll talk about in a bit.
RNA polymerase III makes the tRNAs and the 5S ribosomal RNAs.
These are the smallest RNAs except for these guys over here.
So these are relatively small RNAs, tRNAs and 5S ribosomal RNA.
Now these are specialized.
And these have specialized promoters
that are specific for the systems of transcribing
each one of these.
And we'll see there are, in fact,
systems that are involved in doing that.
What you see on the screen is an extraordinarily potent poison.
It kills a fair number of people every year.
It's called alpha-amanitin, and it's made by certain mushrooms
known as death caps.
alpha-amanitin is exquisitely poisonous
because what it does is it inhibits all of the RNA polymerases
in a eukaryote.
And most importantly, RNA polymerase II,
which makes the messenger RNAs, is very sensitive to it.
A little bit of this, and you're in trouble.
Every year, there are hundreds of, I shouldn't say hundreds,
but there are dozens of people who die
from having eaten what they thought were safe mushrooms,
and they've gotten ahold of some death caps.
If you ingest death cap mushrooms,
you have a matter of a few hours
in which to get a liver transplant.
That's your only hope.
Your only hope.
You've got a few hours.
Now you know how long transplant lists are.
You know, and medical professionals,
what's involved in doing something like that.
That's your window to survive.
Be careful the mushrooms that you eat.
So alpha-amanitin is actually used in the laboratory
as a way of studying transcription,
partly because it is so very effective at what it does.
There's the source.
And I don't know mushrooms well enough
that I could tell that from another one myself,
so I worry about that.
When we look at the transcription in eukaryotes,
I said that we had different systems.
We have a system for an RNA polymerase I,
we have a system for an RNA polymerase II,
and we have a system for an RNA polymerase III.
No, I'm not going to make you memorize this figure
but I do want to point out some general features of it to you.
We're going to focus most of our attention right here
on RNA polymerase II.
And RNA polymerase II, what we discover is that there are,
what we call, control elements.
When you hear the word "element," we're not talking about boron,
or silicon, or something like that.
In eukaryotic cells, we're talking about specific sequences.
Now these specific sequences play roles
in regulating transcription.
The situation in eukaryotic cells, as I said,
is much more complicated than prokaryotic cells.
A promoter pretty much takes care of things
in a prokaryotic cell.
In a eukaryotic cell, we have to have multiple elements there.
Why does it have to be so complex?
Well, because we are so complex.
We only have about ten times as many genes as E. coli does.
Only about ten times as many genes as E. coli does.
But think of the different types of cells
that we have in our body.
We have bone cells.
We have muscle cells.
We have skin cells.
We have pancreas cells.
You name it.
We have different specialized cells.
And the way that those cells are different from each other
is a function of the proteins that are in them.
We don't want my bone cells to be making things
that my muscle cells would be using, for example.
There will be some we want.
Others we don't.
So the mixing and matching, the pattern of proteins
that I want in any given cell, is going to be very complicated.
It's not that for E. coli.
E. coli has to do the following.
"A sugar here.
"Let's break it down and divide."
That's basically what E. coli has to decide.
For a eukaryotic cell, "Am I a bone cell?
"Am I a bonehead?
"What am I?
"What type of cell am I?"
These are very important things.
And so it takes a very complicated system to control that.
We're going to scratch the surface of that.
Do the same things exist for RNA polymerase I?
And for RNA polymerase III?
Well, not quite as complicated.
But we discover that, nonetheless, they have different elements,
in this case promoter elements, that help to regulate
how much of these given genes are made.
That sort of makes sense if you think about it.
You say, "Well, don't we have a constant need for ribosomal RNA
"or a constant need for transfer RNA?"
And it kind of depends on the cell.
Some of the cells in our body are very quiescent.
They don't do much.
They don't have as many needs.
No reason to waste energy on making things
that you're not going to need.
Other cells in our body are dividing very rapidly.
Our intestinal cells that I've talked about will do that.
So we have to think about that in terms of the bigger picture.
Eukaryotic cells also have TATA boxes.
The TATA box wasn't something that was unique to E. coli.
The TATA box in eukaryotic cells
is somewhat different than E. coli.
The sequence is very similar, TATAAAA,
which is where the TATA actually comes from.
This shows the percentage of the time
each one of these appears at this region.
Now the different thing about eukaryotic cells
is the TATA box is not set at one specific place.
In E. coli, it's about -10.
In eukaryotic cells, it can be anywhere from about -35
up to about -100.
It'd be quite a ways away.
Nonetheless, there's a TATA box.
It's not an absolute thing in eukaryotic cells.
Most prokaryotic cells have a TATA box.
You saw some examples where they didn't.
But most prokaryotic cells, their genes have TATA boxes.
Genes that are made in high quantities in eukaryotic cells
will in fact have a TATA box.
So a TATA box is there.
A TATA box plays an important role
in getting the transcription process started.
Now that's probably a good place to do this.
That kind of relieved everything, didn't it?
You guys want to join me?
[Transcription by Kevin Ahern to the tune of Frosty the Snowman]
Are the bonds of RNA
That support a ribopolymer
Made of G, C, U, and A
Though RNA polymerase
Binds to a TATA box
And copies from the template strand
All the along the way it walks
Of transcription thus proceeds
From the closed to open complexing
In the DNA it reads
The sigma factor gets released
Its work is over fast
Polymerase can then advance
After this step has been passed
The polymerizing spree
Moves along the way in fits and starts
Synthesizing five to three
The RNA is floppy and
It dangles from one end
Oh that's a most important thing
For you to comprehend
Fin-ish-ES the RNAs
Thanks to protein rho or hairpin forms
That release polymerase
So this describes transcription's steps
In three part harmonies
Here's hoping with this melody
You can learn it all with ease
Odd place to stop, isn't it?
[Kevin Ahern whistling Frosty the Snowman]