In this presentation we begin to
get an idea of the complexity of RNA
and also the complexity in
the ways that RNAs are made.
I would be covering the topics
here of eukaryotic gene expression
and RNAs and RNA polymerase, as we shall see.
Now in eukaryotes the scenario
for making RNA is very complicated.
The proteins that cover the
DNA making up the
protein-DNA-complex called chromatin
have a major role in basically
hindering gene expression.
So in order for genes to be
able to be expressing eukaryotes,
they have to not only be able to the help the
polymerase find the promoter,
they also have to be able open up the
region so that promoter can be
found and that's what I wanna
talk a little bit about
here. So the complexity is enormous and
moreover the sequences are larger
and the genes are very widely spaced apart.
So chromatin to define it is a complex
of DNA and histone proteins
that make up what we call the chromosomes.
Now the histone proteins are
positively charged small
proteins that the DNA wraps
with it, we shall see.
In order to help the DNA polymerase
to find the right sequence,
proteins called transcription factors
help facilitate this process.
And they work in a variety of ways and
one of the ways I am going to discuss
here is through the use of enhancer
sequences that they bind to, to help
set up the transcriptional machinery.
Now chromatin the first thing I need to
discuss; because, chromatin is really a complex
that has to be dealt with
in order for RNA to be made.
As I said, it's a complex of DNA
protein and in some cases actually RNA
comprising the eukaryotic chromosomes.
For RNA polymerase to perform this
transcription, this chromatin must be
changed so the access can be gained to the DNA.
Now to give you an idea of this complexity, I have
presented these images below and they start on the left
with a very far out view of chromosomes
as might be seen in the microscope.
And then sequentially I zoom in closer, and closer
and closer ultimately getting to the individual DNAs.
So if we start on the far left we can see
what are called metaphase chromosomes where
we have just the basic coils that are
there and these will be visible
in a visible like microscope.
Zooming at a little bit future, we can see these coils
have some various things that help hold them together
and moving even further we get to an
interphase chromosome where we can see
a part of the chromosome that
might be involved in making RNA.
As we look closer, we see the loops
of the individual chromatin
fibers becoming apparent.
The fibers have a structure called the
30nm fiber that you can see here
from a distance and zooming in even
closer you can now see that they have
so very tight organization that's there.
An actively transcribing set of
genes will have a DNA region
that's described as beads on a string
sort of like what you see here.
And examining those beads on a string closer
we discovered that they are
made up of DNA coiled around
these individual histone
proteins that I have described.
And, of course, if we peel away the histone
proteins we are left with paired DNA.
Now a nucleosome is a term that
we need to be familiar with
that looped structure that I described on
the last figure is called a nucleosome.
So it's the simplest unit of chromatin
structure and you can see it here.
Within this loop you see various
colored proteins and there are,
in fact, 8 proteins found within that loop.
There are two copies each of
4 histone proteins called H2a,
H2b, H3 and H4.
The DNA is wrapped around this core as you see here
and then there is an additional protein
called H1 that is on the outside
and you can actually see
this wrapping occurring here.
Now you will notice that the
proteins are positively charged
and that helps them to interact with the DNA;
because, the DNA backbone is negatively charged.
We can also think of the histone H1 is kind of sealing
and holding this structure together.
It helps to stabilize the nucleosome.
Now an important point that I mentioned
is that the histone proteins
are positively charged. This means that
they are rich in the basic amino acids
typically arginine and lysine. And in order to
change the structure of the chromatin we
have to change those positively charged;
because, that's strong attraction between the positive
and the negative is what holds them together very tightly.
And tight access of these
histone proteins to the DNA
actually inhibits the process of transcription.
So we can imagine now that this tight
core has to be sort of loosened up
or given access to the transcriptional machinery
so the transcription can happen.
Chemical modifications actually
happen that allow these changes
to occur in transcription and the
chemical modifications that occur
change the positive charges.
There is a group of enzymes called the
histone acetyl transferases or HATs
that used acetyl-CoA to cover up
some of the positive charges
of the lysine residues that are in histones.
Now this has the affect of neutralizing
their negative charge; because, by
putting these acetyl groups on there.
The charge of the side chain of lysine changes
from being positive to being charged 0.
Now this 0 charge, as you might imagine, allows
for a sort of a loosening of the interaction
that's between the histone proteins and the DNA.
This sort of loosening interaction we cal remodeling
or restructuring of the chromatin and it's necessary
for that to happen in order
for transcription to occur.
So we can see the duplex below round
around the histones loosening up
with the chemical modification so that
we get to the scenario on the right
where now we have instead of many coils
together we have individual coils
like those beads on a
string that we saw earlier.
The acetylated histones in
addition to having their charged
changed and allowing this
opening up, as you see here,
the acetylated histones can
also be a specific target
for proteins that effect transcription;
because, not only is the DNA open but if
that acetyl group is a target for a protein
it's helping to focus the transcriptional machinery
to come to the place where transcription should occur.
Now, histone acetylation favors
what we call euchromatin.
So, euchromatin is a portion of the overall
chromatin that is the part of the chromosome
that is transcriptionally active
so acetylation favors transcription.
We can see this difference here at this
schematic at the lower right of the screen.
We see what's called heterochromatin which was
the structure that we had before the acetylation
and we see the euchromatin
which is what we have after it.
Euchromatin is described as active and
heterochromatin is described as silent.
favors the formation of euchromatin
then removal of the acetyl group is essential
for the formation of heterochromatin.
So there are enzymes called histone
de-acetylases that reverse the effects.
And they do that by removing the acetyl
groups from the side chains of lysine.
Now the lysine becomes positively
charged and this ordered structure
called heterochromatin forms.
That heterochromatin will not
be transcriptionally active.
So in eukaryotes the ability to make RNA
is a function of whether the chromatin is in the
euchromatin state or the heterochromatin state.
Now there are numerous modifications that are made to
histone proteins. I don't wanna leave the impression
that acetylation is the only one that happens. In fact
in a figure I will show in a minute. There is
a lot of different things that can be happened
in individual histone proteins.
These include acetylation and de-acetylation;
Methylation and demethylation,
that is putting methyl groups on,
phosphorylation and dephosphorylation
So each of these individual modifications
can affect the individual histone proteins.
Now this talk I won't be able to go through all of the
different scenarios that are there but you could imagine
that these modifications are there to help facilitate
either the heterochromatin state or the euchromatin state.
The chemical modification to bases in DNA can also
affect this process and we will see
how that comes up in a later slide.
So this slide, in this very complex slide, you
can relax I am not gonna take you through it
But this very complex slide shows
for each of the individual proteins in the histone core,
that is that core of 8 proteins in the center of nucleosome,
you can see the individual modifications
that can happen to each of those proteins.
Now eukaryotes are very complex.
They need a lot of controls.
They are different from E-Coli
E-Coli has some pretty simple needs.
Do I have energy? Do I need energy?
Do I have lactose? Do I need lactose?
In eukaryotic cells there are fine levels of
control that have to be maintained.
Am I a skin cell? Am I a muscle
cell? Am I differentiating?
Will I become something else?
What are my needs right now?
So very very widely ranging needs
and these widely ranging needs are very
important to be controlled properly.
So like we saw in another presentation, controlling
the lactose operon of E-Coli under very simple circumstance
the number of circumstances that a eukaryotic
cell finds itself in are amazingly complex
and that's where we see the complexity
of these controls on the screen.