Lecture 5: Transcriptional Regulation 3
- Next lecture we’re going to be looking at eucaryotic transcription regulation. Today
we compare prokaryotic and eucaryotic transcription regulation.
- Last Lecture: Last time we looked at the components of genetic switches: small
DNA motifs and moved onto protein motifs that interact with the small DNA motif
that together comprise genetic switches.
- This lecture: We’re going to see how these switches function in vivo.
- Last lecture: You can document interaction between DNA and protein in vivo using
chromatinimmunoprecipitation protocol. Today we see how these work in vivo.
- We looked at the interaction between protein and DNA – the genetic switch in vivo.
Let’s take a look and think about how they might have to work in vivo to fulfill their
purpose of turning genes on and off.
- Simple organisms to begin with: prokaryotes. They have simple development with
one single cell type, they can become an elaborated cell form but overall simple cells.
These cells must respond to fluctuations in the environment & perhaps no greater
fluctuation exists than that in resources that are available for the organism to grow
and survive. As a consequence one of the things that occurs is that these
microorganisms/procaryotes are incredibly motile, they can move through media to
search/savage out resources they need to grow divide and reproduce asexually
making progeny daughter cells.
- Movie: the cells are moving rapidly through medium to bind to those particular
resources. The end result is a colony that has grown out and scavenged effectively for
resources in the medium in which it is growing, foraging for resources.
- What they must do is to respond to environmental cues and to the resources that are
available to them and respond appropriately so either to move in the proper direction
to get those resources or to modify their metabolism to acquire those resources and
use them when they become available. They all depend on changes in gene
- Bacteria have evolved a very simple system for responding to environmental cues.
You have a nice simple transcriptional model, there is no nucleus so signals can
move freely to the DNA invoking transcription and then translation – very simple
regulation of gene expression.
- This is simplified further by clustering groups of genes with similar functions or
that function in a sequential manner, for example in a metabolite utilizing pathway or
a metabolite biosynthetic pathway, organized so that the coding sequences are all
under the control of one simple DNA switch and this is known as an operon.
Single RNA molecule
- An operon is a stretch of DNA found on a bacterial chromosome where you have a
group of contiguous genes (coding sequences all in a row) that are transcribed
simultaneously into a single RNA molecule under the control of a single promoter.
- Example of a trp repressor, the trp operon functioning together and we’re going to
look at the trp operon.
- Here we have it illustrated schematically so this is a segment of the E. Coli genome
and you can imagine the chromosome continuing on to make up that large circular
chromosome that makes up the E. Coli genome.
- What we have in a row as promised is a contiguous stretch of genes, coding
sequences for genes E, D, C, B and A. We can see that they are all under the control
of one simple genetic switch that resides upstream of these coding sequences. The
promoter itself comprises of many different motifs of operator sequences and other
small gene regulatory motifs we won’t focus on today. We will focus on the operator site which mainly functions as an on/off switch for the particular operon, allowing
the transcription of those coding sequences. Produced into 1 RNA that is
polysystronic in that it has a number of systrons or coding sequences arranged
adjacent to each other in one contiguous stretch of RNA. What happens is that the
polysystronic RNA is translated into the 5 gene products – the gene products are used
in the biosynthesis of the amino acid tryptophan hence the name trp operon.
Ligand binding determines interaction with DNA
- The way this operon functions is to be responsive to the availability of Trp in the
growth medium or in the environment surrounding the E. Coli.
- In conditions where concentration of Trp is low, there is a protein which normally
functions to repress the activity of the operon that is unable to bind to the operator
sequence. Think of the operator as the switch and the trp repressor is the gene
regulatory protein that turns that operon off. Under low concentrations of Trp, it is
unable to bind, unable to repress the activity of the operon.
- In the presence of Trp, this binds to the trp repressor and as a consequence the trp
repressor binds to the operator site so it's the binding of the ligand of Trp that
determines the interaction between trp repressor and the operator site. The small
molecule/ligand is what determines the interaction between the trp repressor and the
operator site, its DNA target, so ligand binding determines the interaction with DNA
- You shouldn’t think of it just as an on switch but also as an off switch. If the Trp
concentration was to drop again, there wouldn’t be Trp around for the repressor to
bind to so the repressor wouldn’t bind to the corresponding operator site.
- That is how this simple switch works: ligand there, repressor binds, ligand isn’t
there, repressor doesn’t bind. When Trp is present, the repressor binds and you don’t
need to make Trp biosynthetic enzymes at that particular point in time and it's perfect
but when Trp isn’t around, the repressor no longer binds and you get the transcription
of downstream targets and you’re able to make Trp.
- High concentration of tryptophan, repressor is bound and as a consequence RNA
polymerase (blue blob) is unable to initiate transcription so transcription is
correspondingly off & you get no synthesis of tryptophan as a consequence. You
don’t need to synthesize tryptophan when you have it around.
- By contrast when tryptophan concentrations are low the repressor is unbound and
the RNA polymerase is able to bind to the promoter initiating transcription so it's on,
and a cell which is now not having tryptophan available from environment, will
synthesize it on its own.
- This process in which Trp is absent so you now have transcription being on is
called a system of de-repression. So repressed in presence of the ligand and de-
repressed in the absence of the ligand.
- We can have what is known in the first instance as negative regulation and that is
when a bound repressor protein prevents transcription so it is negatively regulating
the gene in question.
- Here in the first example we have a bound repressor protein and in the presence of
an inducer which can also be seen as a de-repressor it binds to the corresponding
Regualtory protein = can also repressor and causes it to move away from the DNA. The ligand switches the gene
called repressor/activator on by removing a repressor – this is de-repression and that is precisely what we saw
Ligand = acts as substrate with the trp operon and the trp repressor is this negative regulation involving
- By contrast, you can have what is known as a co-repressor binding together with
repressor and the binding of this particular ligand allows the gene regulatory protein to bind to the DNA resulting in co-repression, they’re working together to repress the
gene so ligand removal in this instance switches the gene on by removing the
(I think he made a mistake because co-repression is when the ligand which is
tryptophan joins with the repressor to repress the activity of the gene, it's not a de
repression, please let me know if he announces anything about it)
- In both of these instances what we have are examples of negative regulation, the
binding of repressors that are influenced by the presence or absence of the
corresponding ligand to which they bind.
Activation / deactivation
- By contrast, we can have as opposed to repression/de-repression, we can have
activation/deactivation as a common theme. This is where the interaction between a
ligand and a DNA binding protein resulting in the activation or deactivation. This is
referred to as positive regulation because it involves the switching on of genes by the
binding on of gene regulatory proteins
- Here what we have is an activator protein that promotes transcription as opposed to
a repressor which represses transcription.
- Let’s take a look at these examples right here, we have inactivator ligand in the first
one that normally the activator protein is able to bind to the promoter and promote
transcription but in the presence of an inactivator protein, this positive gene
regulatory protein no longer is able to bind so the ligand binds to remove the
regulatory protein from the DNA and switches the gene off by removing the activator
and this is deactivation.
- Alternatively we can have the ligand functioning as an inducer. In this instance, the
gene regulatory protein binds only in the presence of the inducer and ligand removal
switches the gene off by removing the activator. Or to switch it on the other way to
view it is it functions as a co-activator.
- So how to distinguish between the two slides? Negative regulation involves
repression proteins, those switch genes off and positive regulation involves activator
proteins, things that will switch the gene on. The positive regulation versus negative
regulation can be influenced one way or another by either co-repressors/co-
activators, de-repressors/deactivators (inactivators).
- How do these function in a real context?
- We will move away from how ligands function to regulate prokaryotic gene
expression to look at an example or two to effectively further flesh out these switches
by looking at how some proteins can function as both activators and repressors.
- In this particular instance, size does matter – there is a context dependent role and
that is the distance between where the protein has bound and where the binding site
for RNA polymerase is, is very important to whether the protein is going to function
as an activator or repressor.
- So with lambda repressor, if we have one promoter, if the distance is appropriate
distance between operator and promoter binding site, transcription can occur so the
distance between the operator & promoter allow the lambda repressor to promote
RNA polymerase binding.
- By contrast at another promoter, the lambda repressor actually functions as a
repressor where the distance between the operator site and the promoter site is such
that it inhibits the binding of RNA polymerase and therefore prevents downstream
transcription of this particular gene.
- Note that the distance in the first is a good one allowing the recruitment of RNA
polymerase and the other instance, it actually inhibits the binding of RNA
- This is very simple to either recruit or inhibit the ability of RNA polymerase to do
its job to transcribe the downstream sequences. - If you look at this figure in the textbook 7.38, you will find that he’s done
something where he’s made sure that he is showing transcription as occurring off the
right hand side, but in fact in the textbook they have this in the reverse direction and
the reason he showed it this way is how these genes function in vivo, that is the
lambda repressor binds at one site and one promoter goes in one direction and when
it's bound it allows promotion of transcription in this direction and prevents it in the
- There are conditions we will talk about that cause the lambda repressor not to bind
but then allow the transcription of the gene that goes off to the left hand side.
- We have seen a bit of this like in the trp repressor now we will look at this in vivo
for an operon that is with the lac operon.
- We’ve seen this before: it is a stereotypical operon with a promoter and an operator
site as we’ve seen in previous examples. It has 3 different polysystronic coding
sequences Z, Y and A that are going to be transcribed into a polysystronic message
and then translated into 3 different proteins. These different proteins allow, in this
instance E. Coli, to use lactose as a carbon source.
- The proteins are listed in the slide.
- The beta galactosidase hydrolyzes lactose – it breaks the beta 1-4 glucoside/
galactoside linkage that occurs between galactose and glucose so we have the beta 1-
4 galactoside linkage and that is cleaved by the beta galactosidase which gives rise to
galactose and glucose which can each be used as downstream carbon sources
- That was lac Z.
- Lac Y encodes lac permease and this allows for transport of lactose into bacterial
cells so when lactose is present in the medium, lac permease allows the cells to
import it so that it can then be acted on by beta galactosidase to give rise to those
utilizable C sources.
- It turns out that the way in which the operon works is such that the concentration of
lactose is what functions to alter transcription.
- We can use the beta galactosidase activity as a measure of how much transcription
is taking place at the lac operon. If you count the products of beta galactosidase per
cell, we take a look at those per cell and in the presence of glucose, there is very low
production of beta galactosidase. In the presence of glucose and lactose, there is a
little bit of production but in the presence of lactose alone and the absence of
glucose, you get a lot of production of the particular enzyme.
- How does this operon function? - This is shown in the textbook as fig 7.39 and what it shows is that it involves
important elements: the upstream promoter and a binding site that is located nearby
to the operator, the binding of particular proteins (cAMP protein), binding of
- What he wants to do is to break this down and see what’s occurring step by step.
Binds to the operator
- Lac repressor binds to the operator site – in doing so it prevents RNA polymerase
from binding to the promoter. As a consequence only small amounts of lac mRNA
are produced and we saw that there was a small amount of beta galactosidase activity
even in the presence of glucose. So it is a bit leaky and that is important actually.
Binds to the lac repressor
- The presence of lactose, because there is a small amount of beta galactosidase
produced, you have, first of all, the binding of lactose and the binding of a
stereoisomer of lactose allolactose to the lac repressor.
- Allolactose is a stereoisomer of lactose and it also binds to the lac repressor.
Binds to the lac repressor
- It is very important that even in the absence of lactose, there is a small amount of
permease and a small amount of beta galactosidase, the permease allows import of
even just a small amount of lactose into the cell.
- The small import allows for the isomerization of the lactose into allolactose and the
subsequent binding to the lac repressor which is no longer able to bind to the operator
site. When this happens, the RNA polymerase can transcribe the lac operon but only
- There is another component and this is the CAP protein – the catabolite activator
protein seen in the previous lecture and it contains stereotypical the helix turn helix
motif that is able to bind to a given sequence in the DNA.
- What he wants to do is to take a look at what happens to the catabolite activator
protein and why it's called that and how it's functioning to regulate the lac operon. - We saw this slide already but what we haven’t looked at is simultaneously there is a
molecule called cAMP that is produced in the absence of glucose due to catabolic
- The concentration of cAMP is contingent on whether catabolism has taken place or
not so in the presence of glucose there are very low amounts of cAMP and in the
presence of glucose and lactose again low amounts of cAMP made but in the
presence of lactose alone, high quantities of this protein made. This is important
because it influences how catabolite activator protein (CAP) functions.
- Turns out that in addition to the operator site there is also a place where the CAP
can bind and that is the CAP binding site indicated with a C.
- Binding at the CAP requires the presence of cAMP. Without it, CAP can’t bind to
the CAP binding site – so when glucose is absent, cAMP is produced as we saw in
the lactose only conditions and as we can see here what occurs is as a consequence
that CAP binds to the CAP binding site. RNA polymerase is then recruited and away
it goes to generate the downstream components.
- CAP facilitates RNA polymerase binding to the promoter and allows transcription
to take place. So when glucose is absent, cAMP levels go up and transcription takes
- By contrast when glucose is present, cAMP levels go down and we no longer have
binding of CAP to the CAP binding site and low levels of transcription.
- The lac repressor and the CAP working together.
- In the presence of glucose and lactose what we have is no repressor binding but no
cAMP present and as a consequence, no CAP binding.
- In this point in time, the operon is effectively off but weakly on – very small
transcription taking place.
- In the presence of glucose and the absence of lactose the lac repressor is able to
bind but no cAMP present so no CAP binding so the operon is truly in the off state.
- When glucose and lactose are both absent we have repressor binding, cAMP
present so the CAP is able to bind but because the repressor is bound, the RNA
polymerase shown in grey is unable to do its job. The operon is said to be in the off
state, truly off due to presence of repressor.
- Finally in the absence of glucose but in the presence of lactose, we have an ideal
- cAMP is present so CAP is able to bind, allolactose is present so in this instance,
the lac repressor is unable to bind & therefore RNA polymerase is able to be
recruited. The operon is then on and away it goes doing its job transcribing the
polysystronic message – what a great system based only on ligand binding.
- Greatly regulated, 2 proteins (repressor and CAP), 2 ligands (cAMP and
allolactose) and nice simple switch that gets turned on and off contingent on what the
environmental conditions are. - They cou