Lecture 6: Eukaryotic Transcriptional Regulation I
Slide 1 - Today we talk about proteins involved in chromatin. We have a shopping
list of crap to go through, revisit prokaryotic and eucaryotic transcription,
talk about activator proteins. We aren’t actually going to get the latter part
and that will be in the next lecture.
Slide 3 Is packaged into chromatin
Is unable to initiate transcription on its own
Act at a distance residing
- Now he comes back and makes this statement telling the truth, peeling
away the onion and seeing the complexity that resides. There are 5 different
points that underlie differences and complexity between bacteria and
eucaryotic gene regulation. There are actually 6 different points but in the
textbook they only mention 5, the sixth one is the nucleus so that’s another
possibility for regulation and that’s an important point.
- Point number 3 is probably something that is evident to us, there is a lot of
DNA in eucaryotes. We know there are a # of contexts under which many
genes must function – where a gene must be on in your lung at some time
during development and then in your brain at another point in development.
One of his favorite examples is housekeeping genes in animals function
throughout the body giving rise to enzymes that have housekeeping functions
like enzymes involved in glycolysis. Some of those same proteins are
transcribed at a huge amount resulting in a huge amount being made and they
make up the lens of your eye. There must be differential levels of regulation
and must occur in different cell types so it implies that the switches that
reside next to a gene that say transcribe here or transcribe this amount, must
be pretty large at least to bacterial sequences. This is point 3, eucaryotic gene
regulatory sequences act at a distance, thousands of nucleotides away from
the minimal promoter sometimes therefore many possible regulatory
sequences can reside in between.
Slide 4 - The first is that we have nuclear import as a possibility from gene
regulation, not talked about in the textbook b/c it’s separated nuclear import
from gene regulation. The first one is that once you get into the nucleus, you
have chromatin so eucaryotic DNA is packaged into chromatin and as we
seeing the remainder of the lecture that provides regulation opportunities that
aren’t available to bacteria. Because of the way gene regulatory proteins
function, the sequence specific DNA binding proteins bind at specific
regions, read the DNA sequence, bind to these motifs and they affect the
histone code themselves. It is these sequence specific DNA binding proteins
and their interaction that is going to remodel chromatin, an opportunity that
just doesn’t exist for bacterial gene regulation.
Slide 5 - Point number 2 is that eucaryotic RNA polymerase II which makes RNA is
unable to initiate transcription on its own, it's a wuss and can’t do its own
job, needs helpers and we know that from Professor Chang’s lectures. We
know RNA polymerase II needs a set of general transcription factors and
therefore we have multiple steps available depending on which transcription
factors are there, the rate at which they are recruited, the rate at which they
get retained in the complex will determine ultimately the ability to initiate
and sustain transcription.
- This is going back to Professor Chang’s lectures: remember there are many
rate limiting steps not the least of which is that enzymatic reaction are
catalyzed by components of basal transcription machinery and as an
example; the phosphorylation of polymerase II by transcription factor 2H and
this coupled with the availability of those transcription factors and the ability to recruit them is going to provide as the slide says.
- A number of rate limiting steps that can function as transcription control
mechanisms. He wants to underline the fact that it can control the timing and
localization of initiation, the extent to which expression occurs (lot of
transcription or a little) and the duration to which it is sustained.
- The importance of assembling everything together is point number 2.
Slide 6 Control gene expression from a distance
- Let’s take a look at the implications related to point 3.
- This serves an important function not only to provide a number of switches
near a coding sequence, doesn’t have to be upstream, it also ensures that
protein-protein interactions can occur by having things at a distance b/c DNA
isn’t perfectly bendy, it is flexible but not great. If you tether two proteins to
a stretch of DNA shown by the white circle and the red circle, and the DNA
is shown in red is that red line. If we tether those two proteins having them
between 500 to 5kbases apart, they can interact with a high degree of
frequency because DNA is that bendy.
- This is shown nicely in the upper and bottom panels. The top panel shows 2
proteins tethered to DNA and the blue cloud that is around it indicates the
frequency of interaction – can see that there is a high degree of interaction
right adjacent to each other in this cloud. If we tether the two proteins only
100 base pairs apart, there seems to be a zone of exclusion there because
DNA can’t bend back that tightly on itself.
- This can be captured in a graph showing things drop off precipitously
certainly under a 100 ties apart, then there’s an optimal length around 750
nucleotides apart where proteins can interact with each other with a high
degree of frequency.
- So binding of proteins to different sites on DNA will cause interaction
frequencies to increase as you reach this optimal distance. So the intensity of
the blue cloud indicates the likelihood of collision and the frequency of
collision is increased down to 100 bp after which collisions are restricted.
- Also multiples of ten base pairs are important and why? They are important
because they place them on the same side on the double helix, putting them
in the major groove. We tend to find that spaces between binding sites are in
multiples of ten.
- We have the capacity for proteins to bind at a distance that function to
regulate the function of RNA polymerase II at the minimal promoter.
Slide 7 Control gene expression from a distance
- Captured beautifully in this slide from the old textbook and we can see the
binding at a gene regulatory site by an activator protein and we see the gene
expression is controlled from a distance that can be hundreds to thousands of
base pairs away from that minimal promoter where the TATA box is.
- The role that it's playing in this regard is to recruit additional proteins to the
minimal promoter to actually activate gene expression.
- What are the sequences that are there?
Slide 8 Promoter and regulatory DNA sequence
Upstream and downstream of coding sequences
Gene binding proteins
- These gene regulatory sequences comprise: the minimal promoter +
additional gene regulator sequences.
- We’ve learned about the minimal promoter already (TATA box &
surrounding plot) that allow RNA polymerase to bind and do its job.
- But we need in addition to that are sites that recruit gene regulatory
proteins. Most of them are upstream of the minimal promoter but holy crap
look there’s one downstream too, not only downstream of the TATA box but
actually downstream of the whole coding sequence. This tells us that gene
regulatory sequences can reside both upstream and downstream of the coding sequence.
- How could that be true? How can you have gene regulation occurring
backwards? It's always 5’ to 3’. How can you have gene regulatory sites
downstream and functioning in a promoting capacity? Remember DNA is
arranged in a 3D structure and chromatin, something we think of as linear for
downstream, may in fact in terms of chromatin be right next door. The gene
regulatory protein may actually be right beside it and what we want to do is
to change the local structure of chromatin around there so RNA polymerase
can get on and do its job.
- Not only is it downstream or upstream, it can be in introns as well. Introns
(junk DNA) can have regulatory gene sequences there, it can function in
important/crucial gene regulatory role as we will see in the next lecture.
- All of these are target binding sites for gene binding proteins.
Movie - We’re looking inside a cell – zoom into nucleus, look at DNA and a
minimal promoter to start with. TATA binding proteins bind to TATA box.
DNA is bending, & very slowly it is going to recruit basal transcription
factors and eventually RNA polymerase II. Eventually the DNA will unwind
slowly. Eventually RNA polymerase will do its job.
- This is a very simplified version of what’s truly happens b/c what need next
door are activator proteins binding elsewhere in the DNA and they will
eventually recruit the TATA binding proteins. The subsidiary transcription
factors come and here we go again.
- This takes us to the end of point 3.
Slide 9 - Recall that eucaryotic genes are not organized into operons – they are
individual genes, gene regulatory region, coding sequence and that’s how
they function. They can be co-regulated, that is they are transcribed at the
same time but they are never located right next to each other to create a
polysystronic message – no operons so independent regulation.
- Let’s take a look at those gene regulatory proteins that do the job.
Slide 10 Modular
- He wants to focus on eucaryotic gene activator proteins. The same is a little
true for repressor proteins but some themes are different.
- Tend to be modular in structure – this means they have different modules,
all one protein, 2 different bits to the module like having an arm up there and
a leg down there. They are all part of the whole but perform different
- For transcriptional activator proteins, they have a DNA binding domain that
binds to a recognition site and a transcriptional activation domain. These two
domains can be completely separated from each other and their functions
- Let’s show an experiment in the bottom two panels. What we’ve got in this
example is the Gal4 protein with its DNA binding domain which can bind to
a recognition site, the activation domain is able to activate transcription at the
minimal promoter and turn on a particular reporter gene, lac Z in this
- If we take the transcriptional activation domain and create a fusion protein
with a different DNA binding domain, it no longer is able to bind to its
binding site and obviously doesn’t turn the corresponding downstream gene
- If by contrast we fuse it again to a different DNA binding domain but then
make that DNA binding domain available on a stretch of DNA upstream of a
minimal promoter, the transcriptional activation activity is able to be
recruited to a new site, the one bound by lac A in this instance, activate gene
transcription and turn on the lac Z gene.
Slide 11 - So what is it doing when it's doing that? That activation domain is
increasing relatively weak and nonspecific interactions between the DNA and RNA polymerase holoenzyme. It's grabbing the RNA polymerase
holoenzyme and saying you need to be here. It increases it up to a thousand
fold, that interaction. It can do this by recruiting specific members of the
basal transcription machinery and a really important protein complex that is
your blank on the next slide.
- It can do this by recruiting the mediator protein complex. The mediator
protein complex is a multi-protein complex, 20 different proteins that
function to assemble the pre-initiation complex of RNA polymerase II at the
- That’s shown nicely in two slides.
Slide 12 The mediator
- This is the figure from last year: here is the transcriptional activation
protein bound to its activation site with the activation domain and it will
recruit through protein-protein interactions, the mediator which then
assembles the RNA polymerase II transcriptional apparatus at the minimal
- This year, it shows 4 different protein complexes coordinating the mediator
at the same site. It shows how all these different gene regulatory sequences
can reinforce the interaction of RNA polymerase II with the minimal
Slide 13 - What else is going on at that point in time? Recruitment of our old friends,
the chromatin remodeling complex which can increase accessibility to the
TATA box just by remodeling the nucleosomes. It can remove the
nucleosomes altogether resulting in great interaction with the TATA box. It
can change the histones that are present in the nucleosomes again increasing
the interactions with TATA box and finally it can recruit histone modifying
enzymes that can modify the histone code.
- That is our focus for the final point.
Slide 14 Lysine
- This takes us back to our histone code. Recall there are multiple sites for
post transcriptional modification along the amino terminal tail. Recall that
histones can be acetylated on their amino terminal tails – on the lysine
residues making them more neutral. And resulting (and he doesn’t like the
textbook’s saying) in repelling of DNA from the histone proteins – it's not
really repelling, it's just not associating as well so it's looser not really
pushing apart per say, it's just not as tightly bound. The ultimate result is that
you end up with an alteration of the histone code resulting in gene
- He wants to walk through an example in the last bit.
Slide 15 - Imagine we have a nucleosome, we recruit a DNA binding protein to it, and
that DNA binding protein can do a # of things itself – recruit rewriter
proteins, recruit acetyl transferase, recruit histone kinase, adding acetyl
groups and phosphate groups to the histone which are then recognized by
chromatin remodeling reader proteins, TFIID & we end up with initiation of
Slide 16 - This is a flow chart of what he’s been talking about.
- What happens with tra