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Kevin Ahern: ...for coming and subbing for me.
Any comments, questions, concerns,
problems, thoughts?
Everybody's shaking their head no.
Does that mean its good or bad, I don't know?
How was the exam?
Ugh?
Student: As expected.
Kevin Ahern: As expected.
Alright.
How many thought it was a hard exam?
I'm not going to ask those who didn't think it was a hard
exam because your friends would kill you,
so I try to get a gauge of it from those who say that.
So how many thought it was harder than the first exam?
That's almost all, alright.
So a lot more material than the first exam.
Thoughts or comments at all?
You're ready to get in the last nine lectures of the year?
You realize we have nine lectures counting today?
You're supposed to be sad at this!
[laughter]
[class murmuring]
Flunk him from the day.
[laughter]
I get kind of teary eyed just thinking about it!
I mean, you know...
Alright, so we have a fair amount to cover in that time
and a couple students have asked me,
"Do we get notecards this term like
we got notecards last term," and the answer is no.
[class griping]
Oh, the answer is yes.
Alright, alright.
[Kevin laughing]
This is mob rule.
If you guys all decided you were all going to get A's
I don't know what I would do.
I'd be in probably even bigger trouble.
Alright.
Okay, a lot of noise here.
Keep it down so people around you can hear.
So last time when I finished
I was still talking about DNA repair.
I'm only going to say a couple of things about DNA repair.
I will only say a couple things about DNA recombination,
and then we're going to turn
our attention to transcription.
And transcription is obviously the next piece
of the central dogma that we'll talk about.
So DNA repair is, as I hope I convinced you, important.
You might think it's not that important
because DNA polymerase is really very good
at doing what it does,
but there are two considerations here.
One consideration of course is that if you can make a good
editor a better editor then the quality
of your publication, in this case the DNA
that's being produced, is improved.
So the repair system's are very good
and important for that purpose.
And the second is that not everything
that affects DNA affects replication.
So if I have chemical damage or I have physical damage
that occurs to DNA like thymine dimers
that result from UV light exposure,
then having mechanisms in place to repair those
are very very important.
I say that because that may not have been that apparent
from the things I first told you about but there are
human diseases that are clearly linked to problems
relating to DNA repair.
And so I want to say a couple of words about those diseases
since many of you are premedical students
or preveterinary students as the case may be,
and understanding the link between DNA repair
and disease is a very important one.
The first of these I want to mention that don't have a slide
for you is called Huntington's disease.
Huntington's disease is an extremely
debilitating disease.
It affects not a giant number of people
but a large number of people.
It's an inherited disease and it is passed from generation
to generation by virtue of the fact that the manifestations
of the disease don't typically appear until a person
is in about their thirties, forties,
after the reproductive years have largely been exhausted.
So this gets passed because people do have these
characteristics that they pass on.
Probably the most famous person to have suffered from
Huntington's disease was the folk singer *** Guthrie
and he died of the disease in his fifties,
I want to say, and had a famous son named Arlo Guthrie.
It is a recessive trait and悠'm sorry,
it is a dominant trait, not a recessive trait.
It is a dominant trait and so his son had a fifty-fifty
chance of receiving the bad gene from his father.
And it wasn't until, because at the time that Arlo
was reaching the age of maturity there weren't
genetic tests like we have today
where we can examine the DNA and determine
if you have the bad gene or the good gene,
so he didn't know what his life expectancy
was going to be, and it turned out he was lucky.
He got the good gene.
So that was useful.
Well has does that relate to DNA repair?
The deficiency in Huntington's disease arises from
an inability of the replication system to deal with
what are called repeated sequences.
So a repeated sequence can be something like
GCA/GCA/GCA/GCA kind of like we think of with a telomere,
but there are places where repeated sequences appear
inside the coding regions for proteins.
Now what I'm going to describe happens,
it can happen in telomeres.
And if it happens in telomeres
the consequences are fairly small.
If it happens in the coding region for a protein
the consequences can be very large,
and that's one of the reasons that Huntington's disease
is a deadly disease.
So what happens in Huntington's disease is
there are what are called,
these are called triplet repeats.
They're three-base sequences, like I said, GCA/GCA/GCA.
A deficiency of proteins that are involved in the repair
process that stop the what's called the proliferation of
these repeats gives rise to the severe problems
associated with the disease.
What happens if we think about a repeated sequence?
I've got GCA/GCA/GCA.
On the bottom strand I've got what?
CGT/CGT/CGT, right?
So we can imagine that a GCA that's up here on this part
will be paired with an appropriate sequence
on the bottom strand.
But that same sequence is present three base pairs away.
So what can happen during the replication process
is remember the strands are pulled apart.
The strands, when they come back together,
what if they come back in the wrong way
so that this one on the top is paired with this sequence
three bases away or this one six bases away?
Well what you create when you do that is you create a looped
out sequence and now you have two strands
that do not have the same number of repeats in them.
What can happen to that over time is you can change
the number of repeats that exist in that given sequence.
Well since these occur in protein coding regions,
if this is a protein that's involved in the nervous system,
which some of the Huntington's disease problems
have proteins in this regard,
then what happens is now you've changed
the amino acid sequence of a critical protein.
Instead of having maybe three copies of an alanine,
alanine, alanine, you might have fifty.
Well you can imagine based on what we've talked about
in class about how amino acid sequence
is critical for protein structure,
if I change a protein from having three alanines in a row
to having fifty because this thing just keeps changing
and changing, there are going to be some severe
consequences that arise.
And that's what happens in Huntington's disease.
It is a deficiency in some of the genes
involved in DNA repair and gives rise
to some very severe problems.
There are cancers also that arise from deficiencies
in DNA repair.
HNPCC is one, also called Lynch syndrome.
It's an inherited form of a disease
that is linked to colon cancer.
This disease is deficient in several different ways
in which proteins involved in DNA repair
are not properly functioning.
Dr. Andrew Buermeyer on our campus is an expert in this.
His lab researches this and tries to characterize
at the protein level and also at the DNA level
what happens in this mutational mechanism that arises.
If damage to DNA that occurs in,
particularly in this case in the colon,
is not repaired we could imagine that
there will be mutation of genes.
More mutation of course is more likely to affect
an oncogene and more likely to give rise to a tumor.
Another important gene to think about with respect to
DNA repair does not actively play a role in repairing DNA
but it plays a role in the process of repairing DNA.
Well what does that mean?
Well p53 I'm sure many of you have heard of by now,
but if you haven't I'll tell you a little bit about it.
p53 is a gene that is involved in what's called
quality control with respect to DNA replication.
There's a gene that is found in virtually all
eukaryotic cells, or equivalents of it thereof
are spread throughout eukaryotes,
and what it does is it monitors the replication process.
Monitors the replication process so that葉he replication
process you may recall I said in eukaryotic cells
is fairly carefully orchestrated.
It occurs during a specific part in the cell cycle.
And there has to be some assurance
that everything is complete,
that is replication has completed and it has completed
properly before the next phase of the cell cycle occurs.
We can't have cells starting to divide if the DNA
is not completely replicated.
So p53 plays a very important role
in basically telling the cell yes,
replication is complete and everything's okay.
Well replication doesn't always go okay
and if there are problems that occur during the replication
of the chromosomes p53 actually steps in.
So if there's let's say chemical damage that has happened
to a segment of a chromosome and the DNA polymerase is going
along and it can't deal with that damage
that happens to be at the replication fork,
that chromosome will not be able to complete replication.
When that happens p53 literally steps in
and stimulates activation, transcription,
translation of enzymes involved in DNA repair.
So p53 acts as a transcription factor,
that it is it can activate the transcription
of other genes.
And it's invoked when the signal arrives that replication
has not proceeded properly, has not completed properly.
At that point p53, because it steps in and stimulates
this repair process and stimulates the activation
of these genes, stops the cell cycle in its tracks.
So that's the physical stop that happens.
The cell cycle does not proceed
and the cell is basically given time
to try to repair that damage.
So the genes involved in the repair process
get synthesized, they travel to the place
where the damage or where the replication has stalled,
and they act to try to repair that damage.
There's two possibilities, of course.
One is that they repair the damage
and if they repair the damage then p53 says,
"Okay, I'm going to let go of the cell cycle
"and let the cell cycle proceed
"so that the cell can go ahead
"with the division that it was already setting out to do
"with the synthesis of the DNA."
The other possibility, of course, is that p53悠'm sorry,
that the repair process may not complete.
Repair enzymes throw up their hands and say,
"There's no way that we can fix this."
When that happens p53 initiates a process called
cellular suicide, also known as apoptosis.
And that's spelled A-P-O-P-T-O-S-I-S,
the most mispronounced name molecular biology.
The second P is silent.
"Ap-uh-toe-siss."
And when that happens the cell commits suicide.
There are a series of events that happen
that basically result in the cell dying
and not proliferating and passing on what would be
bad genes to daughter cells that would of course
have a high potential for having mutations
in important genes.
So p53 is very very critical player in this process.
Not surprisingly, some cancers arise because of mutations in p53.
So imagine that General Motors has a quality control
division that inspects every car before it comes off
the assembly line and occasionally finds a car
that has a dent in it or a car that has a nonfunctioning
engine or something.
That quality control group would obviously take that car
away and not allow it to be sold.
p53's doing a similar thing.
Take away that quality control group
and you're going to have more cars
that are going to slip out the door
that are going to have problems and you're going to have
some very unhappy consumers at some point.
So not surprinsingly if we take away that quality control
of p53 we're going to have some cells
that are going to have some problems.
Okay.
So that's in a nutshell some of the important things
to consider with respect to the proper repair
of damage to DNA.
These two things on the screen
(I'm only going to talk about the one on the bottom)
are important affectors of DNA.
They can bind to it.
They can covalently bind to DNA and basically stop
DNA replication or cause damage
that would otherwise be problematic.
One of these, the one you see on the bottom called cisplatin
is actually used in a variety of chemotherapy approaches.
Cisplatin can, as I say, bind to DNA,
prevent its replication, and if the target is a tumor cell
we would imagine that this would be a very useful tool.
Alright.
The last thing I'll mention about DNA repair
is that there is a standard laboratory test
that is given to try to determine what we describe
as the mutagenicity of a given compound.
The mutagenicity.
What does mutagenicity mean?
Well mutagenicity means the likelihood to favor mutation.
The likelihood to favor mutation.
Well you hear about compounds and you say,
"Oh, that's a carcinogen."
That's a carcinogen, well it's linked to cancer.
It would be useful to know if I have a mutagen
because a mutagen is probably itself
going to be carcinogenic because
it's going to favor mutation.
You've already seen that the more mutation you have
the more likely you'll have damaged cells
that lead to problems down the line.
Well there's a test called the Ames Test
that is used to measure the mutagenicity of compounds.
So I want to just spend a minute and tell you
a little bit about that.
The Ames Test can be performed fairly simply.
It's not a perfect test but it's a pretty darn good one
and what's beautiful about it is its simplicity.
It relies on the fact that it directly allows a researcher
to compare how many mutations happen in the presence
of a given compound compared to the absence
of that compound.
That's what it allows a person to do.
Now this figure unfortunately is not the best figure
for describing what I'm going to tell you.
I show it because that's what your book has
but I don't have a better figure to show you
so I'm going to have to tell you a little bit about this.
In the Ames test, if I take a plasmid
and the plasmid has the coding for a given gene,
that gene might be, let's say,
the ability to produce a blue color.
If I had that plasmid and I put it into a bacterium
the plasmid will make a blue color, right?
Well let's say I'm interested in studying how
mutagenic this compound is.
Imagine I put this plasmid into a bacterium
and I treat it with this chemical that I'm interested
in studying and I ask the question,
"Does it affect how much blue color I get?"
Well that would be one way of doing this experiment
but it wouldn't be a very effective one
because most cells aren't going to be affected
by this compound.
I need something that's more sensitive than that.
What if I get 3,000,124 cells that have blue color
and I get 3,000,000 cells that have blue color
in the treated one?
I say, "Well I've got fewer cells that have it,
"so is it mutagenic?"
Well that isn't a very sensitive assay because
that's within my experimental error,
3 million versus 3.1 million.
So what I do is I flip it around.
I take that same gene and I make a single mutation
in the gene that's in that plasmid.
One single mutation but it's a very critical one
because when that mutation is present no gene is made.
No blue color.
So I take this plasmid and I give it to cells
and I have a bunch of cells that have no blue color.
They've got the plasmid
but they've got this defective gene.
Everybody with me?
Bunch of cells, defective gene.
Now, I'm interested in studying things
that favor mutation.
With this system I've just described to you
I can study cells, how frequent they mutate
that one critical base.
And how will I know?
Well every time it mutates that one critical base,
what's going to happen?
Blue color, right?
So to do my experiment I take my cells that already have
the plasmid with the defective gene
and I divide them in half.
One half gets the treatment.
Let's say I'm interested in studying saccharine.
Is saccharine mutagenic?
One batch of cells gets saccharine.
One batch of cells doesn't get saccharine.
I plate them out on plates and I ask the question,
"Which one gives more blue color?"
Will some cells have blue color
even if they don't have saccharine?
Yes.
Why?
Because like that famous bumper sticker,
"Mutation Happens."
It'd be a great bumper sticker, wouldn't it?
Mutation happens.
It happens at a certain frequency.
When I plate cells I can put several million cells
on a plate, and if I have a mutation rate that's let's say
one in a million, I'm going to have a few of those colonies
that are going to come up that are going to have blue color.
But the beauty of this method is that I can say,
"Well how many blue colonies do I get in the presence of
"saccharine compared to the absence of saccharine?"
If the presence of saccharine has fifty colonies
and the absence of saccharine has one colony, hmm.
Maybe saccharine is mutagenic.
Maybe that's why I'm seeing that increase in numbers.
This is a very simple test.
It's not proof, but it's a very simple test
that allows us to answer the question,
"Is this compound mutagenic,
"at least in E. coli?"
Is it mutagenic in human cells?
That's another matter.
But this is the basis of the Ames Test.
Alright.
Now I'm doing all the talking.
I will slow down and take any questions
that you might have about that.
Yes, Connie?
Student: With the Ames Test, [inaudible].
Kevin Ahern: So for the Ames Test does that very
specific mutation have to happen
for that blue color to be produced?
Yes because it was a specific mutation
that stopped it from being produced in the first place.
Student: [Inaudible].
Kevin Ahern: Does it undercount the number of mutations?
I'm not trying to count the total number of mutations.
I'm trying to compare the number of mutations.
It's relative.
So I have the same parameters in each set of cells,
so it's a relative number that I'm getting.
I'm not claiming that the Ames Test,
and that's a very important point.
It's a very important point of confusion.
People think, "It tells me how many mutations."
No it doesn't.
It tells you the relative number of mutations.
Make sense?
Jodie?
Student: So this is primarily to test for
single base pair mutations, right?
Kevin Ahern: This is testing for the likelihood
to make a single base pair mutation, yes.
Student: What about excisions or frameshift?
Kevin Ahern: Again it's a simple test.
Excisions and frameshifts will affect that
and they may give rise to blue color.
So it's not exclusively that but the design
is for single base pair mutations, yes.
As I say it's not a comprehensive test.
But if I'm really interested in the compound,
it's very easy for me to test and see, well,
in the Ames Test is it getting me a higher number
of blue colonies?
That's useful.
Okay.
Oh, yeah?
Student: [Inaudible].
Kevin Ahern: Is Huntington's disease found
in other animals or just humans?
That's a good question.
I believe it's found in other animals as well
but I don't know,
I'm not an expert on Huntington's disease.
I don't know that but I would be surprised
if there wasn't an animal equivalent of it
since it's a very general reproductive failure.
So the last thing I want to talk about
very briefly is recombination.
And recombination, we hear about it.
We don't really study it an awful lot,
at least not on this campus.
Many people have their whole lives
dedicated to studying recombination.
But it's a very important process.
It's important to recognize
that recombination allows segments of DNA to get swapped.
Not everything that happens in the cellular division process
arises solely from replication.
So there's other forces that can drive mutation
besides replication processes,
and one of these is recombination.
So recombination allows different DNAs to swap segments.
Cells have built-in mechanisms to allow this.
Why in the world would a cell be favoring a process
that's allowing mutation and change to occur?
The answer to that question is if it didn't occur,
we wouldn't be here!
We've selected for it.
We have selected for it.
We see proteins that facilitate recombination
starting all the way in the very simplest of organisms
and proceeding all the way through human beings.
With recombination it is safe to say
that you have segments of DNA in you
that neither of your parents have.
Neither of your parents have.
We think about, "Well I get one chromosome from Mom
"and one chromosome from Dad,"
and therefore everything I have is like Mom and Dad.
But segments of those chromosomes can mix and match
on the way to getting to you and as a consequence you have,
every person has, DNAs that are not identical to DNAs
in either of your parents.
It's for that reason that in some cases
identical twins may not be absolutely identical.
Mutation can also affect that.
But recombination is a darn good way that a very tiny segment
of DNA can be present in one twin but not the other
depending on when the recombination event occurred.
Well the mechanisms of recombination are really more
than I want to go through here
but I will just briefly show you this method here
called strand invasion, and strand invasion happens
when DNAs of same or similar sequences align with each other.
This can happen during the replication process.
And when this happens, if we look at, let's say,
my two identical DNAs here, it's not unreasonable
that if I take a piece of the top strand here
-or let's say the bottom.
I guess the bottom strands will get used
so the bottom strand here- and I let it invade a partner DNA
that there's going to be quite a few,
maybe not perfect base pairs here
but quite a few base pairs here because again
they're related sequences to start off with.
Proteins in our cells will facilitate this.
They facilitate this because, again, over evolutionary time
it's biologically favorable to allow this process to occur.
Well once this guy invades there's got to be resolution
and there's resolution that happens as a result
of something that's called a Holliday junction.
This shows that invasion process.
And no you're not going to have to regurgitate this
because I couldn't draw that if I had to.
But a Holliday junction is an intermediate that's formed
during recombination
that involves what's called a cruciform structure.
It looks like a cross.
There's an X that's there.
Well this, if you follow it all the way through,
involves strand invasion.
Strand invasion may be followed by replication.
It may be followed by repair.
And because of these processes, especially the repair process,
the mismatch repair that's there,
now we create some new sequences that were not present
in either of these DNAs to start.
This can allow proteins to evolve.
This can allow proteins to gain new function.
This can cause problems.
But this is yet another mechanism
whereby this change can occur.
Ultimately this guy has to, these two sequences
have to resolve the each other, and the product of this
gives two DNAs that have different sequences
than either one of them had to start with.
This process is called homologous recombination
because it requires the sequences
to be very similar to each other in start.
Not all recombination is homologous recombination.
Not all types of recombination are homologous recombination.
There are some systems that appear
to insert DNAs relatively randomly.
One of the cellular proteins that's involved
in resolving this process is called integrase.
I-N-T-E-G-R-A-S-E.
There are systems that have integrases
and one of the systems that has an integrase
that's very similar to the ones that we have in our cell is ***.
***, as part of it's life cycle, has to insert itself
into human chromosomes in order to make a stable infection.
Needless to say, one of the strategies for stopping
the proliferation of *** is to inhibit the integrase,
and there are some drugs that are out there that will do that.
Now, ***'s integrase is a really good example
of one that does not do homologous recombination.
It inserts relatively randomly.
So we can't predict where *** is going to insert
in a human chromosome.
It's going to go in many potential places,
it may go in many places.
And that's one of the issues that arise
in trying to deal with it.
That's what I want to say about recombination.
Comments, questions?
Glen?
Student: So you said [inaudible]...
Kevin Ahern: I'm sorry, say it again.
Student: You said that some genes
are not from our parents in our bodies,
[inaudible]?
Kevin Ahern: Okay, so his question relates to the fact
that I said that you have sequences in you
that are not identical those of either of your parents.
These are relatively few, but imagine if you will that,
and his question then is does that have a minor
or major impact?
It depends on the sequence.
So there'll be some sequences that may recombine
and when they recombine make a nonfunctional gene.
If that's a nonfunctional gene
that's critical for survival, gone.
You never see them, right?
That gene might have to do with
a slight lightening of the hair.
That gene might have to do with something
we couldn't even physically measure.
So there's no set answer to that.
It really depends on the nature
and the diversity of genes that are out there.
But recombination can happen with any gene,
any gene in the genome.
Well let's turn our attention to transcription.
We're moving from DNA to RNA.
And transcription I will talk about in this class
in two respects.
So what I'm going to talk about today and on Wednesday,
and probably it'll now spill over into Friday a little bit,
is transcription in a very general sense.
I'll talk about translation after that
and then I'm going to come back
and I'm going to talk about transcription
as part of a bigger process called gene expression.
So you're going to get transcription largely in two doses.
The first dose is going to be fairly basic,
probably things that you've covered in other biology classes.
The gene expression component will probably be in more depth
and may not have been things that you've heard of before.
Well, I always warn my students that you darn sure
should know what the term transcription means.
Am I saying you should know what it means,
it means that you should have something
besides a definition of it in mind.
You've got to pound into your head that transcription
occurs when RNA polymerase copies a DNA and makes an RNA.
I will warn you about this.
I will ask you a question on the exam about this
and I will guarantee you that at least a quarter of you
in here will get it wrong.
I've warned you.
DNA replication, transcription, translation.
You should have no confusion whatsoever
about what those involve.
You should have no confusion whatsoever
in terms of what's being made and how it's being made.
Absolutely you need to understand that.
Transcription as I said, synthesis of RNA from DNA
requires an enzyme called an RNA polymerase.
RNA polymerases are similar to DNA polymerases,
at least in some respects.
If we look at RNA polymerases
we see that sort of hand structure.
Now the hand is turned sort of like this.
The DNA polymerase had that hand
and we said that the double helix fit inside of here,
those fingers were sort of holding it, supporting it,
and we see something like that as we look at RNA polymerases.
Moreover, if we compare prokaryotic verses eukaryotic
we see again structure is not very different
from one to the other.
Structure implies function.
A structure is important for function,
therefore structure will be conserved
if it is an important function,
and yeah it's absolutely an important function.
RNA polymerases differ from DNA polymerases
in several respects.
First, RNA polymerases do not, underline not,
require a primer.
DNA polymerases require a primer.
Complete difference between the two.
There are many similarities between RNA polymerases
and DNA polymerases.
Both work in the 5' to 3' direction.
Here's the difference.
RNA polymerase doesn't work nearly as fast
as DNA polymerase does.
DNA polymerase will kick along
at about a thousand nucleotides a second,
at least in E. coli.
In E. coli RNA polymerase moves along
at about fifty nucleotides a second.
Why the big difference?
Well one of the reasons is that,
and this is another difference,
RNA polymerases don't make long stretches of nucleic acid.
A few thousand base pairs
is as much as an RNA polymerase will make.
It's not a race.
As we will see in prokaryotes, the synthesis of RNA
and the synthesis of protein occur in the same time and place.
And so RNA polymerase's rate of movement in prokaryotic cells
appears to be about the same rate of movement
of the synthesis of the protein on that RNA.
So that coordination between the two
may be very, very important.
RNA polymerase only copies one strand.
DNA polymerase of course copies both strands.
There's leading strand and lagging strand replication
in DNA synthesis but there's not in RNA synthesis.
Only one strand is copied.
DNA replication I told you started at origins.
RNA synthesis also occurs at specific places.
They're called promoters.
Not surprisingly, a very common feature of promoters
are A/T rich sequences.
Part of the transcription process
requires the strands being separated
in order to get the RNA polymerase in,
and the easier it is to separate those strands,
the easier it is to start RNA synthesis.
Your book goes through talking about subunits.
I'm not going to talk about subunits
and I'm not going to hold you responsible for subunits.
They do have some interesting separate functions
but I'm not going to talk about them here.
I do want to think a little bit about
copying of a DNA to make RNA.
We remember of course that another difference
is that in RNA we use UTP instead of dTTP.
I think everybody knows that.
So RNA will have U's in it where DNA had T's in it.
Now, here's a DNA sequence that's here in red and blue.
Here is the RNA that's made from it.
Which strand was copied, the red or the blue?
The red was the copied strand
because I have A where there was T, an A where there was T.
Think about this.
The other strand is going to have the same sequence
as the RNA does.
It's going to have the same sequence as the RNA does
except it's going to have T's in place of U's.
We call the two-we give names to the two strands of DNA
relative to the RNA.
The strand that's being copied or has been copied
is called the template strand because it provided a template
for making the RNA.
The strand that has essentially the same sequence
as the RNA is called the coding strand.
Now, another feature of this figure
that I want you to recognize is right here where this bar is.
With RNA we invoke a numbering system
because that numbering system helps us to orient
where in the DNA the RNA is being made.
You'll notice that +1 is the very first nucleotide
where the RNA is made.
There's +1, there's +2, there's +3, +4, +5,
et cetera, et cetera.
If we go away from where that start point was
we give them negative numbers.
There's no zero in this scheme, by the way.
Now we'll see that the negative numbers
will be important as we describe control sequences
known as promoters because the promoters are located over here
in the negative numbers for the most part.
I don't want to talk about that.
And I don't want to talk about that.
And I don't want to talk about that.
What this figure is alluding to is something
I'll just briefly mention to you
and that is that RNA polymerase doesn't move as fast
as DNA polymerase does, and it moves
in what we call fits and starts.
It'll go for a stretch and then it'll slow down.
It might even back up, and then it'll go forwards,
and then it'll sort of back up.
And this might seem a little confusing
but it appears that there's two factors at play here.
One factor is that the RNA synthesis system
is not set up like the DNA synthesis system is.
In the DNA synthesis system we had a helicase
that was unwinding and pulling everything apart
as rapidly as it could.
We had a topoisomerase system that was setting up
and was not allowing those strands to get untangled.
RNA polymerase doesn't have as sophisticated
of a system in which it works.
That means, then, that RNA polymerase
is going to be more affected by the sequences
it is moving through during the transcription process.
Because remember it's moving between
those two strands and moving down.
It's moving in what we call a bubble.
Those strands are peeled apart.
It's copying one strand,
the other strand is just going the other direction.
That means that sequence will affect
the rate with which it moves.
You might expect that G/C rich sequences,
harder to pull apart, might slow down the process.
They do.
The other thing that's important with respect to the movement
is this moving forwards and then shuffling backwards
appears to be helping the RNA polymerase
to do at least a little bit of proofreading.
A little bit.
The synthesis of RNA is much more error prone
than the synthesis of DNA.
It's going slower and it's more error prone.
What gives with that?
Well again, speed is not the most important thing
and fidelity is not the most important thing either,
fidelity meaning the accuracy with which it is copying things.
Well why not?
Remember, RNA is not passed from generation
to generation to generation.
In fact what you will see with RNA is once it's made
in the cell it has a lifetime.
It gets made, it gets used, it gets broken down.
And there's a cycle of that.
It gets made, it gets used, it gets broken down.
If the cell makes a mistake making a given RNA, not a biggie.
It's going to make fifty others
that aren't going to have that mistake.
It doesn't have that option with DNA.
It doesn't have that option with DNA.
So it's very important then that cells...
let me back up a sec, say it the proper way.
That the synthesis of RNA is not as critical
as the synthesis of DNA is for the lifetime
or the longevity of the organism.
Errors can happen and things will still proceed.
Now some proofreading is done
but not nearly as much is done as there is in DNA.
Let's talk in the time I have left about promoters.
Promoters are easiest to understand starting with prokaryotes
and that's mostly what I'm going to be talking about here.
What is a promoter?
In prokaryotic cells a promoter is different
in terms of functionality than it is in eukaryotic cells.
In prokaryotic cells we can describe a promoter as a sequence
that RNA polymerase binds to and starts transcription nearby.
It's a sequence that RNA polymerase binds to
and starts transcription nearby.
In eukaryotic cells that we'll talk about later
it's much more complicated.
We're going to start with the simplest first.
Now, what you see on the screen is a depiction
of negative numbered sequences relative
to a bunch of different genes in E. coli.
Here's five different genes in E. coli.
And when people line these up for hundreds of genes
they started seeing some characteristic patterns.
At about -10, and this is approximate,
but at about -10 most prokaryotic cells
have what's called a TATA box.
They have a T/A/T/A rich region at about -10.
Now from what I have told you about hydrogen bonds
and so forth this should make very good sense
because the RNA polymerase is going to start over here
having some A/T's to pull apart easily
close to that is really useful.
The TATA box makes very good sense.
This TATA box is also called the Pribnow box, P-R-I-B-N-O-W,
named by the person who first recognized this.
Not all genes have the same sequence.
There is a, in fact as you look across this
there's not a single one that has a TATAAT.
Well why do we call it,
why do we have this sequence down here?
This is the average sequence if we compare hundreds of genes
and we say, "What's the most likely T here?
"What's the most likely the sequence here?
"It's A.
"What's the most likely sequence here?
"It's T."
Not all of these genes have that.
Does that mean that these don't work as promoters?
No.
But it means something very important.
The closer a given sequence for a promoter of a gene is
to a perfect TATA box, the more RNAs will be made from it,
the RNAs starting over here.
So if I have something that's a TATAAT,
the gene that's associated with that,
the cell is going to make a lot of that RNA.
The more RNA it makes, the more protein it makes.
Now we haven't talked about it before
but we have to start thinking about quantitative things.
DNA replication occurs and we get two copies, ***.
We do two and we get four.
But in a given cell, cells have different needs
for different proteins.
You saw when I talked about DNA polymerase III
that there were only five or six copies
in the entire E. coli cell, but for DNA polymerase 1
I said there were thousands.
Part of that control is exerted by how many RNAs are made.
Things that I need more of I make more RNAs of.
So we see that changes in sequences relative to the promoter
will allow a cell to sort of decide
how much of this that I need.
That's really useful.
I don't want to waste making thousands and thousands of copies
of DNA polymerase III if I only need six proteins per cell.
It's a waste of energy.
That cell is not going to be competing very well
because it's going to be wasting its energy on making things
that it's not going to use.
On the other hand, if I need a lot of hexokinase
because I've got all kinds of sugar there
and I'm only making one or two copies of that RNA per cell,
what's going to happen?
Well the cell is not going to have enough hexokinase
and without hexokinase very little glycolysis,
and glycolysis is where the cell gets its energy.
That cell is also going to be dead in the water.
So the level of synthesis of RNA
is a very, very important factor.
We're going to spend a lot of time
talking about it in the next few days.
On that note I will let you go.
[END]