Download 9/18/08 Transcript I

Fundamentals I
9/18/08
Dr. Ryan
10:00-11:00
These slides are numbered based on the NEW powerpoint Dr.
Ryan used in class**
There’s a lot to cover and get right to it.
Slide 2: Eukaryotic RNA polymerase
• I mentioned yesterday that in eukaryotic there are three RNA
polymerases. They are called RNA polymerases. There are three
DNA dependent RNA polymerases: Labeled RNA Pol I, II, and III
 Each of these their own sets of promoters and genes that they
transcribe.
 All 3 are big, multimeric proteins (500-700 kD).
 All have 2 large subunits which are very similar to prokaryotic.
RNA polymerase subunits  and ' which we talked about
yesterday.
 And the catalytic site may be conserved between prokaryotes
and eukaryotes.
 All of these polymerases interact with general transcription
factors-GTFs. WE will go into detail on what they are.
 How do you discriminates between these three? How do you
know if they gene they transfer by Pol I II or III?
• There is drug called -amanitin and Pol II shows the most
sensitivity. This is an inhibitor of pol II. -amanitin is a
bicyclic octapeptide and blocks the elongations.
Slide 3: Lecture Part 3 RNA polymerase II inhibitor -amanitin
 The sensitivity is shown there.
 Pol II is more sensitive than Pol III and Pol I is hardly sensitivity
at all.
 This drug -amanitin comes from the mushroom called the
“destroying angle.” So if you’re picking mushrooms for your
morning omelet, stay away from these.
Slide 4: Yeast RNA polymerase II subunits
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These are the subunits of RNA Pol II from yeast. There is a total
12 subunits.
The nomenclatures is RNA.
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o This is RPB1, that’s for RNA Polymerase. B stands for the
Pol II. RNA Polymerase I would be RPA 1-12. For Pol II
subunits, they are all RPB and then the subunit 1-12.
 The first subunit shares homology with the beta prime subunit of
prokaryotes.
 One of its important features is the C-terminal domain, its
repeating heptapeptide, has lots of serines and theorinines in
it that get phosphorlylated from the process of going through
transcription initiation to the actual start of transcription. We’ll
talk about that later.
 The second subunit and these are labeled 1-12 according to their
size, they discovered or purified these later.
 The second largest subunit RPB2 shares homology with the Beta
subunit of E.coli. It has the NTP binding site and forms half of
the catalytic site with the RPB1.
 There are two subunits that share similarities (Dr. Ryan corrects
himself by saying one shared similarity) with alpha that’s
RPB3.
 The promoter recognition has homology to the sigma factor is
RPB4.
You can see, number of these factors are shared between the three
polymerases: RNA Pol I, II, and III.
Slide 5: Transcription Factors
 In eukaryotes, the transcription factors are going to very
important. All three polymerases interact with transcription
factors.
 A lot of these interactions are protein-protein interaction or
protein-DNA interactions.
 We’ll talk a lot about types of transcription factors and these
general transcription factors used by all these polymerases.
 These transcription factors (TFs) recognize and initiate
transcription at specific promoter sequences. So when 6 billion
base pairs of DNA, these transcription factors are going to help
land the polymerase on the promoter to initiation transcription.
 Some transcription factors bind to specific recognition sequences
within the coding region. So they are not upstream where the
promoter is seen in prokaryotes. Sometimes they bind internally
to the genes.
Slide 6: Helix-Turn-Helix Motif
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I would like to talk about three classes of transcription factors.
These are figures from your book.
They talk about these three classes.
First is the helix-turn-helix motif. These transcription factors
all bind DNA and what’s special about these is the helix-turnhelix motif is shown in orange in these figures.
And these are two alph-helices that are separated by beta turn.
The first helix sits down in the major groove in DNA, the second
one locks in place. You get very specific type of binding. Most of
these helix-turn-helix motif bind has homodimers or
heterodimers.
Yesterday, we talked about DNA having dyad symmetry, having
inverted repeats.
An inaudible question is asked about not having the exact slides.
Answer: These are in the start of Day 4. This is the lecture of combo
of 3rd and 4th lectures. All lectures are in one of these two lectures.
They are mixed.
So just listen carefully. You will have a copy of these available to you.
Another question: paraphrase: Is there a PowerPoint with your notes
that we can get?
A: It’s written in your book. You see when you get a test questions
and you see helix-turn-helix or alpha helix beta turn alpha helix you
will know what it refers to.
Another question: paraphrase: What is important ?
A: I won’t ask you anything that I don’t have on this slide or is not in
your book in the reading material.
Back to lecture:
 Alright, by forming a homodimer or heterodimer, you can
increase the specificity of DNA binding with these factors.
 One dimer binds one sequence or one protein in the homodimer
can bind its cognate sequence on the other side and they have
an interaction domain so you can increase the specificity of
binding of these factors.
 Now we talked about these factors yesterday, some are of these
are prokaryotes and eukaryotes that have these helix-turn-helix.
 Down here, is the cap protein that we talked about.
 The activator protein of the lac operon. It has the helix-turnhelix motif.
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Here is the trp represson and lac repressor that we talked about
yesterday.
Eurkarotic helix-turn-helix motif that is called the antrion (sp?).
o Helix-turn-helix motif is located in the homodomain.
o This is important for DNA binding.
o The specific sequences upstream are very important
developmental genes.
Slide 7: Zinc-Finger Motif : C2H2 Class
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The second class of transcription factors is these Zinc-Finger
transcription factors.
There are 1000s of these in our genome.
The typical structure is a pair of 15 amino acids apart separated
by about 12 AA in the finger, and 2 histidine, 2 or 3 amino acids
apart.
o There is a zinc atom that coordinates these 4 amino acids.
o Now the actual DNA binding part of the zinc-finger is
located in the 12 AA space (between the cystiene and the
histidine).
Here’s the actual structures.
So it’s got an alpha helical structure.
o Each of these fingers can contact 5 base pairs of DNA, and
these fingers always come in 2 or more fingers together.
o So you can have up to 17 fingers in some of these
transcription factors. So if you have multiple fingers they
can bind/interact longer sequences of DNA.
o Now they each interact with 5 base pairs, but they have a
tight interaction with three and interact with a neighboring
one.
o So each finger, if you have 3 fingers are together they
actually interact with specific 9 base pair sequence in the
DNA.
o If you have 4 fingers, you would have 12.
o The middle finger is has interaction with the neighboring.
Each contacts with three specific bases. S
o If you mix and match these fingers- there’s a company do
this- they can build you a transcription factor that will bind
any DNA sequence you want to make- so they say.
So they again they use these fingers, there multiple finger they
can bind to longer and longer stretches of DNA specifically.
Slide 8: Region Leucine Zipper Motif bZip
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The third class I would like to talk about is the basic region
leucine zipper motif (bZIP). These transcription factors are
mostly alpha helical in structure.
They have a basic region that makes contact with DNA.
And the similar to the helix-turn-helix motif and the zinc finger
factors, these bZIP factors also bind in the major groove of DNA.
o All three of these major classes all bind to major groove of
DNA.
Now what’s special about these is that the single factor will
dimerize with another bZIP factor.
o They do this via this alpha helical domain called the
leucine zipper.
o If you turn this alpha helical regions and look at it, look at
the amino acid that are located- every 7th amino acid is a
leucine.
o Now the Leucine all lineup on one face of alpha helix. It’s
called an amiphatic helix. It’s very polar on one surface.
On the surface with leucine, it’s very non-polar. Non-polar
on the other surface. **NOTE: this is what I got out of
what he was trying to say. He had broken thoughts. ***
o See the two leucine surface come together-zip together.
You can picture the zipping action. That’s why they get the
name bZIP.
o So these can bind has homodimer or heterodimers. You
can get two different bZIP coming together.
o Their DNA recognition sequences may be different. So
when they contact the DNA, you can be specific in your
DNA binding.
Slide 9: bZIP transcription factor
 The next figure is from your book showing a heterodimer coming
together and interacting in the major groove of DNA.
 Here is the basic region sitting in the major groove.
 Here is one of the proteins, and there’s the second one behind
the helix sitting in the major groove. This is would be the leucine
region.
 They cut of the rest of the protein which would have activation
regions that will activate transcription.
Again, there’s description in your book for all three of those.
Slide 10: General Transcription Factors
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Now the general transcription factors help position the RNA
Polymerases on transcription initiation sites. (The next few slides
are from the end of Lecture 3)
These general transcription factors help the polymerase come in
and land on the initiation site and form along with the
polymerase a transcription- initiation complex. The nomenclature
is determined by the polymerase that they are associated with.
o So, the TF- transcription factor, the II means it associates/
or its general transcription factor for RNA polymerase II.
o And then various complex of the proteins that have been
isolated, and as they were purified and given letter
designation of TFIIA TFIIB etc.
o We will in more detail in a moment.
So again these help the polymerase associate with the promoter find
the initiation site.
Slide 11: General Transcription Factors
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Of the general transcription factors, TFIID is the largest and
consists of a TATA box binding protein, that is abbreviated TBP.
It has 8-10 transcription TATA box binding protein associated
factors which are abbreviated TAF.
Again the II there for TAFs II are because they are isolated from
TFIID of RNA pol II.
The TFIID is composed of TBP and the associated TAFs. Those
together make up the TFIID.
TBP is a “universal transcription factor.”
• It associates with promoters of all three RNAPs (pol I, II,
III)
• It is also used even though it’s called the TATA box of
binding proteins, it’s also used with promoters that don’t
have a TATA box. These promoters that don’t have a TATA
box, have to rely on other general transcription factors
that help the polymerase find the initiation site.
TFIID has two roles:
• Foundation for the transcriptional Pre-initiation complex.
• Prevents nucleosome stabilization in the promoter region.
• If you have to transcribe a gene, you don’t want
nucleosomes to bind there and wrap it all up. So if it’s
gene that’s expressed all the time, these so called
“housekeeping genes,” you want to have this factor around
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keeping the nucleosome from closing up the promoter
gene.
Slide 12: TBP is used by all 3 RNA polymerases
TBP- TATA binding protein is used by all three polymerases. It has
different names depending on the other proteins its associates with.
 When it’s in a Pol I gene it’s called : SL1 factor.
 When it’s interacting with Pol III, its called Transcription factor
III B.
 We have already talked about Pol II, it’s TFIID.
 It does not always to bind to TATA boxes. Pol I and Pol III, and
sometimes Pol III promoters don’t have TATA boxes. But, TBP is
still used as a general transcription factors.
Slide 13: Yeast TATA binding protein TBP.
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This is diagram of the yeast TATA binding protein. What’s
unusual is that TBP binds in the minor groove. Previous
transcription factors we talked about were all major groove
binders.
This is actually bound in the minor groove, while in binds it
bends the DNA by 120 degrees. It also melts this region. If this
region is usually A-T rich area, it helps melt this region upon
binding. As it binds, it’s got amino acid chains that go in the
minor groove and that helps pry the DNA apart at that region.
I will talk briefly talk about the promoters for Pol I and Pol III and get
back to Pol II.
Slide 14: Promoters of RNAP I
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So the promoters of RNAP I…
Pol I is basically there to transcribe the large ribosomal gene.
Our genome is 100s of similar copies of the rRNA genes.
Remember from yesterday talk, rRNA is the most abundant RNA
in the cells- it’s over 80% of the RNA is rRNA. We have one
polymerase in Eukaryotes that does nothing but synthesize/
transcribe through these genes. There are 100s of them.
The RNAP I promoters are called class I promoters because they
are transcribed by the RNA polymerase I. There are two
elements: upstream control element and a core element.
The two different transcription factor binding sequences: SL1
and UBF.
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We already know that TATA binding protein is a component of
the SL1.
Slide 15: Transcription of rRNA genes by RNA polymerase I
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So this is example of what the rRNA gene looks like. It’s an array
of gene after gene with a spacer in between. The spacer is red.
The transcripts are initially one long precursor transcript about
45s. Out of the transcript, it’s heavily processed to get the 18S
and 5.8S and 28S rRNA.
You will get this more when they talk about translational of
ribosomes. He then asks the class: Have you had a lecture on
ribosomes? It’s coming…
These ribosome are ribonucleoprotiens- they are mixture of
many proteins and RNA, There are the three RNA components18 S associates with 30 proteins to form a 40 subunit, 5.8 S and
28 S .
The 5S, which comes from Pol III transcription, joins with about
50 proteins to make the 60S subunit- a large subunit.
Now third polymerase, Pol III. This polymerase transcribes
various small RNA’s. The : 5S rRNA, tRNAs (15 % of total RNA
in the cell), vary abundant messages.
There are various small nuclear U6 snRNA that are also
transcribed by pol III.
Eukaryotes have specific polymerase that will transcribe tRNA
because of the larges messages.
Slide 16: Promoters for RNAP III
 Genes like the 5S rRNA and tRNA are called class III promoters
because they use RNA polymerase III. Some of the genes
transcribed by Pol III are similar to pol II promoters- two these
are used U6 small nuclear RNA and 7SL RNA genes. But they are
used pol III. They determine this by used alpha amitain to see if
these are sensitive or not. These genes were not.
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The 5S rRNA promoters are entirely within the coding region of
the gene. I will show that on the following slide.
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The tRNA promoters contain two elements.
Slide 17: PIC Assembly for RNA Pol III Genes
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Here’s the tRNA gene and the 5S rRNA gene. There are series of
these general transcription factors. Notice there are called TFIII,
see here- for transcription factor for pol III, complex c. They
load on these box a and b in a particular order forming this preinitiation complex.
The TFIIIB has the TATA binding protein in it. And you will
notice that for the tRNA gene, both of these boxes are located
downstream at the promoter in the sequence of the trNA genes.
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Similar for the 5S ribosomal, RNA gene, there’s a series of
factors that bind TBP is part of TFIIIB again. It helps position
these general transcription factors that help polymerase over the
right startt site for transcription of this chain.
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Question: What is a good thing to take from this slide?
A: TBP is used for all three of polymerases. These binding sites
for these general transcription factors don’t always occur
upstream at the transcription site in these genes. Some of these
TF RNA some sRNA don’t use pol II to trassncibe their genes;
they use pol III.
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Look for the broad picture. GF’s help orient the polymerase in
the right spot to intiated transcription.
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Question again: Can’t here it.
A: The only time I would ask you memorize specific will be for
pol II, that we will talk about. You have the general concept, if I
have what TFIIIB to bind Pol II gene, that’s not correct
If I have TFIID is used as general transcription factor for tRNA gene
transcription is incorrect. For tRNA transcription, is TATA binding
protein utilized? Yes.
Slide 18: RNA polymerase II General Transcription FACTORS
(GTF)
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Let’s switch to pol II. These are the GTF’s for pol II.
The first one of these is listed sort on in the order that they from
the initiation complex. The first factor, TFIID –again it’s
composed of TBP and all the TATA binding protein associated
factors (TAFs). Together, TBP and TAFs make TFIID.
I have the function of these in the next series of slides. We are
going talk about the function as we go. This is in your note, so
you can get basic idea of what they do. We are going to make
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the pre-initiation complex and start transcription in the next
series of slides.
Slide 19: TBP-associated factors (TAFS or TAFII)
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The TAFs or TAFII use the TBP associated factors- these are
important to bind TBP to promoters that lack TATA boxes.
Originally it was thought all genes had TATA boxes, but we are
finding out that majority of genes don’t have TATA boxes once
you analyze them.
There are different TAFs in different cells.
So these TAFs complexes were isolated from cell type. If you
look at a different cell type, the complexes that you isolated
from the GTFs could be slightly different or very different.
In vivo these factors are associated with additional proteins
forming a larger complex of about 50 polypeptides.
So again for every cell in a given particular gene, there are
different times of expression or development, things can be
different.
It is hypothesized that a single large complex come together
first, assemble, and get loaded on to the promoters.
Question: If it lacks TATA box, how does it work?
Answer: It’s still associated; it brings all these TAF’s. SO there
are associated through the TBP. Some other TAFS can also
interact with DNA. So those TATA-less promoters, those other
TAFs are more important. Or finding the start site of
transcription.
Ok lets make a pre-initiation factor here for pol II transcription.
Slide 20: Eukaryotic RNA Pol II Transcription Formation of PIC
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If this is a gene that has a TATA box, will bind TATA via the TBP
subunit.
The TBP, part of the TFIID, will bring this complex of proteins, so
TBP and TAFs down the TATA box and bind. It will bend the DNA
and binds to the minor groove.
It bends the DNA and melts the two strands apart.
Next the TFIIA stabilizes interaction on the TATA box.
Slide 21: Eukaryotic RNA Pol II Transcription Formation of PIC
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Next, TFIIB which is a monomeric protein comes in and binds.
This kind of helps place the TFIIB, it kind makes contact with the
initiation are the start side of transcription. It helps ground
things- getting ready to for the polymerase to come in the right
location.
TFIIB also interacts with a lot of other transcription factors that
may be binding distal promoters that we will talk about in a
moment – like enhancer like sequences.
So these promoters not only have this massive transcription
factor sitting at the initiation site, but there are also other
transcription factors that are upstream elements and
downstream elements. All these interact to bring in the
polymerase and get efficient transcription.
TFIIB plays a role interacting in some of these other things –
accessory transcription factors.
Slide 22: Eukaryotic RNA Pol II Transcription: Formation of the
PIC
 TFIIF binds to RNAPII, and they make a preformed complex.
This (the TFIIF) helps direct, the RNAPII to the TFII D, A, and B
already setting at the promoter.
 Next the TFIIE binds to the TFIIF/RNAPII complex, and this
further helps to cement that initiation site.
 TFIIE is a DNA-dependent ATPase. This is probably what
generates the energy for the helicase that’s the next step, which
comes in with TFIIH.
 Now I might mention, the Carboxyterminal domain (CTD), so
that’s in the Beta-prime subunit of the RNA polymerase- that’s
the largest subunit. It helps form the catalytic site. It has this
long CTD with all of these amino acids: Ser and Thr that can be
phosphorylated. It has to be in an unphosphorylated state to
come in and bind at this step. Later we’re going to see that it
gets phosphorylated, and that enables transcription elongation.
Slide 23: Eukaryotic RNA Pol II Transcription: Formation of the
PIC
 Once you have Pol II and TFIIF and TFIIE down at the promoterthat activates TFIIH.
 TFIIH now contains nine subunits.
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It has helicase activity, and it’s going to help unwind the
transcription bubble. It needs ATP to do this, and it’s probably
coming from the TFIIE.
This TFIIH also has It has protein kinase activity, and it will
phosphorylate the CTD of the Beta-Prime subunit of the
polymerase.
Once the CTD tail of FNAPII is phosphorylated, it detaches
RNAPII from TFIID, and you get the beginning of transcription
and elongation of the gene.
So this is just a pre-initiation complex. It’s sort of like this is the
basal level of expression. All of these general transcription
factors bringing in polymerase just helps you initiate
transcription.
We haven’t yet talked about all the other factors and enhancerlike proteins and other transcription factors that help stimulate
or repress this process.
Slide 24: Carboxyl-Terminal Domain (CTD Tail)
 The carboxy-terminal domain is a heptomer, so it’s a stretch of 7
amino acids that is repeated many times: Tyr-Ser-Pro-Thr-SerPro-Ser
 In the mouse there’s 52 of these. (Referring to the repeating
sequence above, I think) on the CTD.
 It’s very important for transcription that you delete this. In the
mouse you can make knockouts, you delete that tail- the
animal’s dead.
 Critical for viability
 CTD tail becomes phosphorylated on some of these Ser and Thr
residues, not Tyr- that’s a typo there. Well it can be
hydroxylated on a Tyr too. They have a hydroxyl group.
 This is what allows the polymerase to transcribe away from the
promoter.
Slide 25: RNA Pol II Promoters
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There are other elements in the promoter that also affect the
level of transcription.
We’ve talked about the TATA box bringing down TFIID and the
initiator that’s located right at the +1 transcription start site.
There’s also upstream elements downstream elements. We can
consider these proximal promoter elements. They can be
upstream within the gene or downstream.
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Slide 26: Eukaryotic TATA Box
 This is a schematic figure that is from your book. It’s showing
the eukaryotic TATA Box.
 Again, if you line up a bunch of genes that have TATA boxes,
there’s a consensus sequence that can be derived, showing the
frequency of A’s and T’s at these various positions.
 On average there’s about 25 spaces upstream of the +1
transcription start site.
 Again you’ll notice at transcription start sites there’s normally a
purine.
Slide 27: Eukaryotic Promoter Regions
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Further upstream of the TATA box you’ll find some of these
proximal promoter elements.
They can be arranged… you know the sky’s the limit. Different
genes will have different combinations of factors. Sometimes
there’s a repeating of the same element over and over again.
These are just some of the…. For these three genes, some of the
factors that have been shown to be in the promoters and be
necessary for the basal layer of transcription for these
promoters.
Slide 28: RNA Polymerase II Promoter Consensus Sequences of
Transcription
Factor Binding Sites
 Some of the factors that bind these promoter proximal elements
are shown in this table.
 The CAAT box binds a factor called CTF or NF1.
 The GC box binds a factor called SP1.
 The octomer box binds Oct-1, Oct-2.
 There’s a B box that binds NFB and so on.
 For any particular gene, the factors that bind there depend on
the sequence upstream of the promoter.
 In vitro transcription experiments people have determined which
of these transcription factors are important and which ones
aren’t.
Slide 20 (on old ppt): Synergistic Activation
 Just to nail this point down: these proximal promoter elements
can be upstream- they can be downstream. They’re usually
multiple elements in these important sequences. They can be
considered enhancer-like sequences when they’re downstream
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or far away from the promoter. We’ll talk more about enhancers
in a minute.
Slide 29: Proximal Control Elements of Genes: Modular Factor
Binding Sites
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You can see the same factor can be repeated over and over
again.
In this case, GATA-1, they’re showing a globin gene here, and
the GATA-1 factor binds at sequence GATA.
There’s multiple GATA-1 binding sites, upstream and
downstream of globin genes.
Slide 30: Coordinate Regulation Via Response Elements
 Just like in prokaryotes, sometimes we want to coordinate
expression.
 This is done in eukaryotes with response elements.
 These are transcription factor binding sites again.
 They are specific sequences where you can control a whole set of
genes by upregulating a specific transcription factor.
 Similarly responsive genes will have a DNA sequence located in
cis to the gene called a response element. It’s just a DNA
sequence that this particular factor will bind. This factor can
bind multiple genes to coordinately regulate increase expression
or decrease expression of genes that have these response
elements in their promoters.
Slide 31: Response Elements: Coordinate Regulation
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We’ll show that here. This is a picture from your book showing
the metallothionein gene promoter.
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Here’s the transcription start site, at 0. This is -20, -40, and so
on upstream. Each of these colored boxes are a specific DNA
sequence that binds transcription factors.Here’s the TATA box.
(@ -25)
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Each of these (BLE, GRE, MRE, ect…) are response elements.
Some of these elements are shown here (in the box) with their
name.
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Here is the glucocorticoid response element or GRE. You can
seen in this promoter there’s a GRE upstream from -240 to -270.
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This metallothionein gene would be responsible to the hormone
cortisol. If you have cortisol on cells the glucocorticoid receptor,
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upon binding cortisol will come and set on this GRE element on
the promoter and cause the up-regulation of the metallothionein
gene expression.
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To clarify how response elements work, what they’re for: you
can coordinately regulate numerous genes.
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Again, you can look at the specifics of these different elements in
your book. I’m not going to hold you responsible for knowing
the sequence that each of these bind or anything, but you
should know that a GRE would bind a glucocorticoid
receptor.
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Question from the audience: Do we need to know what the
other ones do?
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Answer from Dr. Ryan: Heat shock elements are, and some of
them are… I will spell them out. I just won’t put GRE. If I say
heat shock element, you know it’s a heat shock protein, so it’s
probably binding there, huh?
Slide 32: Coordinate Regulation by Hormones/Steroid
Receptors
 Question from Dr. Ryan: Have you had a lecture on steroid
hormones?
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Answer from class: Mutterings of no…
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No? Well you can get coordinate regulation by hormones with
their steroid receptors.
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They have a structure. If you line them all up they have a
hormone-binding domain. Usually they have a DNA binding
domain. Then they have a variable region which can be the
activation or repressing domain.
Slide 33: Steroid Receptors
 Let’s stick with the glucocorticoid receptor here, GR.

Here again it has hormone binding that’s going to bind cortisol.
It’s got a DNA binding, and it’s got this variable region.
Slide 34: Activation of Transacting Factors
 This is an example where the glucocorticoid receptor is usually
masked inside the cell, waiting for its hormone signal to activate
transcription.
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
It’s masked by an inhibitory protein that sits and interacts with
the hormone binding domain and the DNA binding domain. It
keeps the DNA binding domain from being accessible for binding
to its glucocorticoid response element.

This masked receptor and inhibitor is sitting it the cytoplasm.

If you dump cortisol on these particular cells, cortisol will go
through the membrane. It’ll bind to the hormone-binding site
here, cause a conformational change that results in the removal
of this inhibitory protein complex.

So this steroid cortisol comes in, the inhibitory protein goes out,
and now you’ve exposed the DNA binding site.

Upon binding hormone this glucocorticoid receptor complex
moves from the cytoplasm to the nucleus, and now the exposed
DNA binding domain can find the gluococorticoid responsive
element.

Now whether it activates or represses transcription depends on
the context of all the other factors binding and sitting on that
promoter of that particular gene.

Ok… there’s…. we’re discovering new ways transription factors
can be activated all the time.

This is just a schematic showing a few ways.

Just by making a transcription factor. You synthesize, you
translate, it’s active, it goes to work.

You can also have ligand-binding which activates. In the case of
the glucocorticoid receptor, this would be an example of ligandbinding.

You can have a protein that gets phosphorylated to become
active. When it’s not phosphorylated, it sets there inactive.

You can add a second unit so you might need a subunit to bind
to it to become active.

You can have mixtures of these things. Here we talked about
again the glucocorticoid receptor where you have an inhibitor.
When that inhibitor comes off it becomes unactive. In the
glucocorticoid receptor, when the inhibitor comes off it helps it
move from the cytoplasm to the nucleus, and it uncovers the
DNA binding domain.
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
There’s also where you get release from a membrane. You have
a proteolytic event that activates a factor, making an active
transcription factor.
Slide 35: Control of Cellular Differentiation By TFs
 Transcription factors can also control cellular differentiation.

This is just a schematic showing in a simple form how this could
work.

If you have an embryonic cell that divides, and in this daughter
cell it induces regulatory protein number 1 here, that makes this
daughter cell a little different from this daughter cell here. This
cell here may start to express 2 and 3. You can see depending
on which of these factors are expressed in particular daughter
cells, they could have a different developmental fate.

Again, this is all done by TF’s.

Different factors binding different sequences can activate the
transcription of different sets of genes in different daughter cells.

So, you can go from an embryonic undifferentiated cell to
multiple different cell types.

This was a hematopoietic stem cell, and by expressing different
TF’s at different times, you can wind up with RBC’s, T-cells, Bcells, NK cells, macrophages and whatnot- all the lineages in the
blood.
Slide 36: Enhancers
 These are Control elements that stimulate transcription.

They usually bind multiple different transcription factors.

These transcription factors usually have activation domains will
interact with general transcription factors to boost polymerase
loading or just increase the amount of transcription at a
promoter.

You can also have enhancers that work in a negative way, and
those are called silencers.
Slide 37: Enhancers
 Enhancers can stimulate expression of genes over great
disances.
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
In the human Beta-globin locus, all the human Beta-like globin
genes are controlled by a master enhancer that is 50kb
upstream of the locus. It controls the high-level expression of
all these downstream genes. How it does this is most probably
by looping, and it comes and interacts so that all the factors
binding this enhancer sequence, the DNA between the enhancer
and the structural genes are looped out, and there’s a proteinprotein interaction.

These enhancers can be upstream, downstream, in introns or in
exons

They normally work in an orientation independent manner. So if
you cloned a piece of DNA that has an enhancer upstream, if you
clip it out, turn it around, and put it back in, it usually works.

These enhancers can be cell-type specific. So, the enhancer
upstream of the globin genes only works in erythroid cells. You
put that same enhancer upstream of some other genes or if you
cut out the whole globin locus and put it in a lymphoid cell, it
doesn’t work. Ok, only in erythroid cells, and that’s because in
erythroid cells you have erythroid-specific TF’s already bound at
the promoter and these proximal elements to increase the
erythroid-specific transcription.
Slide ?: Proximal Regulatory Regions of Genes/ Slide 38:
Enhancers: Action at a Distance
 This is just a schematic to show an enhancer working at a
distance.

Here again, if this is 50kb away from this transcription start site,
there’s DNA in between that’s looped out, this enhancer binding
protein here can interact with the general TF’s that are going to
sit on this promoter and can bring in these general factors and
other receptor proteins such as a mediator and increase
polymerase loading on the promoter and increase transcription.
Slide 39: Insulators / Boundary Elements
 There’s also special elements called insulators or boundary
elements.

Insulator boundary elements have two functions. If this is a
gene B- let’s call this a globin gene again, and this is that
powerful enhancer upstream. As part of the upstream of this
enhancer there’s an insulator element. What this does is that it
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prevents the enhancement of genes on the other side of the
insulator.

So this, (gene A) is not a globin gene.

In the case of the globin locus, these are ordinate receptor
genes, and it keeps these ordinate receptor genes off in
erythroid cells.

These insulator-like elements can also stop the encroachment of
closed chromatin, the heterochromatin form neighboring loci.

In an erythroid cell, this insulator element keeps
heterochromatic regions next to the globin locus from coming in
and shutting down the genes.

This enhancer sequence keeps all this region open, and keeps
these genes expressed at a high level. These insulators block
heterochromatin from coming in, and it also blocks the
enhancement of genes outside of the locus.

There was a question about the insulator and RNA Pol going back
to transcribe DNA which I could not understand. This is what Dr.
Ryan said: Polymerase is going to come in and bind right at this
promoter, and what direction do all polymerases transcribe?
5’3’ Once that polymerase is done transcribing, and the
transcript’s terminated, it will come off, and it can be recycled
right back on the promoter. So, as long as the general
transcription factors are binding here, they’ll bring in more and
more polymerase, and it will keep transcribing. This gene in an
erythroid cell….let’s say this is a globin gene. You get a lot of Pol
II transcripts of the Beta-globin gene, but thos ordinate receptor
gene would not be open and accessible. The erythroid-specific
TF’s don’t have binding sequences. They don’t have the binding
elements upstream of the ordinate receptor genes, so they’re
not going to be bound there. They’re not going to bring in the
TATA binding protein or TFIID. They’re not going to bring in all
those general TF’s because they weren’t meant to be expressed
in this cell. The right milu (sp?) or TF’s aren’t present there.
The right combination of apples, oranges, and bananas aren’t
there for that one, but they’re in there for gene B.

Another question which I couldn’t understand on the audio.
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
Dr. Ryan’s Response: If there were general TF’s sitting here, but
they needed this enhancer to get a high level of expression, this
insulator would block that.
Another indiscernible question or comment.

Dr. Ryan’s Response: Now, if you put an insulator sequence
right here (downstream of the enhancer), then you’re going to
block the enhancement of gene B also. People have used these
insulator sequences to flank genes of interest that they want
expressed in certain areas but not have the flanking sequences
influence their expression. So they’ll flank their gene of interest
with these insulators.
Another question, but I couldn’t make it out on the audio.

Dr. Ryan’s response: Again, these insulator elements work by
binding factors- transcription factors. So there’s a whole bunch
of binding sequences or binding elements that are specific types
of insulator elements.
Question:

Response: Only in erythroid cells is this whole locus open.
Questions:

Response: These insulators aren’t all that common. They’re
typically found in… in the globin locus there’s all of these globin
genes. They’re all regulated the same way- only in erythroid
cells, and the whole locus is flanked by these insulator cells. Not
every individual gene is flanked by these things. Now the whole
region of this chromosome may be flanked. If you have a whole
bunch of housekeeping genes, they may be flanked by these
insulators. Now, this erythroid locus in a non-erythroid cell,
none of this stuff is happening. All of this heterochromatin is
closed up. The proteins that bind this enhancer aren’t around.
The erythroid specific TF’s which opened up this whole locus by
binding to the enhancer and these promoters aren’t there in
non-erythroid cells. Again the type and number of TF’s that are
expressed in any specific cell control all this.
We’re going to finish the lecture today talking about a very
specific example of…oh boy… I believe he skips the next few
slides due to running out of time at this point.
Slide 41: Silencing: Histone Deacetylation HDAC & Slide 36:
Histone Acetylation and Deacetylation
 There’s ways to open and close chromatin. We talked yesterday
about histones and chromatin. There are enzyme complexes
called HATs (Histone Acetyl Transferases) and HDACs (Histone
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Deacetylases) that add acetyl groups or remove acetyl groups
with the deacetylase. This will help open and close the
chromatin.

If you remove this positive charge (from the NH3+ group of Lys)
by putting an acetyl group there, it will help repel neighboring
nucleosomes and help open up the chromatin.
I believe he skipped this next slide(#42).
Slide 43: Histone Code
 This is a very hot topic of research today: the histone code.

On these amino-terminal arms on the various histones, they can
be modified. They can be acetylated, methylated,
phosphorylated, and ubiquitinated.. These modifications have a
profound effect on gene activity. They can increase gene
transcription, or they can silence gene transcription. There’s a
whole code that’s just now being understood that the histone is
not a static thing. It’s very active in setting up which genes are
transcribed in a cell.
Slide 44: Chromatin Remodeling
 Now, the chromatin can be remodeled.

If you’re closed up tight, how do you open it up and express the
Beta-globin gene?

If you have a hemopoetic stem cell that’s making a RBC
progenitor, you have to be able to open up this chromatin and
get these erythroid-specific TF’s in there to bind their promoter
elements and enhancer elements.

So there are complexes of proteins that come in and can help
remodel the DNA and histone complex.

This is an ATP-dependent process.
Slides 45: Gene Activation By Chromatin Remodeling
 This is just a schematic showing a remodeling complex coming in
that can help pry the DNA off the histone, and these TF’s can
come bind, and that will set the stage to bring in more and more
factors and open up the chromatin for gene expression.
Slide 46: HAT’s open chromatin ( Image slide) & 47: Assembly
of preinitiation complex on open chromatin
 Let’s say you have a hemopoietic stem cell again, where the
Beta-globin locus is totally closed. It’s not expressed, and we
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want to turn this into an erythroid cell and express the Betaglobin gene.

So if you’ve got closed chromatin, you need to make an
erythroid-specific TF, it’s going to come in and associate and
make things a little more accessible.

Other HAT’s can come in and recognize this TF and start
acetylating the histone tails, opening up the region even more.

You’re going to have the nucleosome remodelers come in, and
that will make it more accessible for more TF’s that will come
bind these transcription sequences and open things up even
more.

Once you get to a certain point you can start bringing in the
general TF’s and the polymerase and sit at the promoter and
start transcription of the gene.
Slide 48: Chromatin remodeling (Cow–cloning slide)
 The big picture: what happens in development.

We have a single-cell fertilized egg- a zygote that’s going to
make every cell in the body.

I just want to impress upon you the power of these TF’s with this
experiment. You can think about it when you go home tonight.

Cloning: we can clone cows today… right?

In this cloning experiment we take an unfertilized cow egg. We
pull out that nucleus- that haploid nucleus. In this case they’re
using an epithelial cell from the oviduct. This is a cumulus cell
from the oviduct. They take a diploid nucleus from this cell and
put it along side this enucleated egg.

They electrofuse these together, so this diploid nucleus goes into
this egg without a nucleus.

All those proteins: TF’s, chromatin remodelers, all those protein
complexes in there go to work on this incoming epithelial cell
nucleus. Within hours that epithelial cell nucleus is
reprogrammed into a zygote.

All the genes that were on these epithelial cells are shut down,
and all the genes necessary to be a zygote and begin all of
development to produce a calf have to be turned on.
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
This can all happen in hours time.

Mouse cloning is 3-6 hours.

Cow cloning is about the same time frame.

So all these proteins are setting in this egg.

So, all the things we talked about: chromatin remodeling,
HAT’s, HDAC’s, ect… All this stuff is going on to remodel this
epithelial cell nucleus into thinking that it’s a zygote.

Things like this reprogramming are going to be in the news more
and more as we talk about it. We can now take skin cells and
reprogram them into something that looks like an embryonic
stem cell by expressing four factors.

It’s amazing the plasticity there, and it’s all done by these
protein factors.
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