Download Regulation 1. Short term control

8-1
Regulation
Several aspects of regulation have been alluded to or described in detail as we have progressed
through the various sections of the course. These include:
(a) compartmentation: This was not described overtly as a control mechanism, but is implicit
in the situation of β-oxidation of fatty acids (degradation) occuring inside the mitochondria and
synthesis occuring in the cytoplasm. In this way, if one pathway is turned on, there is no
competition from the other pathway operating in the opposite direction.
(b) hormonal: The effect of hormones was mentioned in a few sections, but the main contact
was in glycogen metabolism where adrenalin was shown to activate protein kinase in the
complex process affecting glycogen synthesis and degradation.
(c) feedback inhibition and activation: There were many examples of this ranging from
energy metabolism to pyrimidine synthesis. This is often referred to as short term control (see
below), because the inhibition or activation can occur as fast as an effector molecule can bind to
a protein.
(d) protein modification: This is best illustrated by the role of protein kinase and protein
phosphatase in glycogen metabolism. This is also an example of post-translational control
Other types of control occur at the level of RNA and protein synthesis (transcriptional and
translational controls). Of these, transcriptional control has been found to be more common.
These are often referred to as long term control (see below) because the response time is
longer requiring either the synthesis or degradation of a protein.
(e) control of RNA levels: As noted this control occurs at the level of either transcription or
translation and is referred to as long term control.
1. Short term control
A
B
In a linear pathway, the end
product E acts at an early step,
shutting down the whole
pathway and saving energy.
C
D
E
feedback inhibition
by an end product of
the pathway
Pi
N-carbamoyl
aspartate +
apartate
carbamoyl phosphate
aspartate
transcarbamoylase
CTP
8-2
Whereas control of a linear pathway is relatively straightforward, control of a branched pathway
presents more of a challenge because of the existence of multiple end products (E and G below).
A system is needed to allow both end products a role in regulating overall expression of the
pathway and a number of systems have evolved that make this possible.
A
B
D
E
F
G
C
(a) multiple enzyme control: Multiple alleles of the same enzyme exist to catalyze a step early in
the pathway that are affected differently by different inhibitors. This controls the overall pathway
but additional sites of control are required in each branch after the branch point.
D
E
F
G
I
A
B
C
II
(b) sequential feedback: The end products inhibit a step after the branch which cause a build up
of an intermediate which acts sequentially to inhibit a step early in the pathway.
A
B
D
E
F
G
C
(c) synergistic feedback: two end products work in concert to inhibit a step early in the pathway
and together have a much larger effect than individually. Variations on this include cumulative (the
effect of E and G are additive) and concerted (neither has an effect individually).
95%
10%
A
B
15%
D
E
F
G
C
8-3
We will return to this topic towards the end of the section for examples. One amino acid
biosynthetic pathway will suffice to provide examples of all three types of feed back inhibition in
a branched pathway. However, that same pathway provides an example of how long term
control can modulate a pathway. So we will first consider long term or transcriptional control
mechanisms and then look at the example.
2. Long term control: control of transcription
By controlling transcription, the amount of mRNA encoding a protein or enzyme is regulated which
in turn affects the amount of protein synthesized. Presumably one of the reasons that
transcriptional controls are so common is that it provides for greater energy efficiency in stopping
both RNA and protein synthesis.
Two examples will be provided to illustrate transcriptional control. The first is in the catabolic
(degradative for energy production) lac operon and the second is the anabolic (biosynthetic) trp
operon. The differences inherent in the two operons are a reflection of the metabolic roles of the
operons.
(a) lac operon - negative and positive control of a catabolic operon
An operon is a grouping of genes, the products of which have a common metabolic purpose. In the
case of the lac operon, that purpose is the metabolism of lactose (galactose-β1,4-glucose) or milk
sugar. The operon encodes a transport protein (lactose permease) to transport lactose into the
cell and a cleavage enzyme (β-galactosidase). There is also a third gene encoding β-galactoside
transacetylase presumably required for detoxification, but not important to this discussion.
The key enzyme for this discussion is β-galactosidase which catalyzes two reversible reactions:
lactose
(gal-β1,4-glu
galactose +
glucose
allolactose
(gal-β1,6-glu)
β-galactosidase mRNA
The synthesis of β-galactosidase mRNA is affected by the carbon sources present in the
growth medium and changing them changes the expression.
succinate
repressed
+ lactose
+ glucose
(with lactose)
induced
Time
catabolite
repressed
8-4
The rationale explaining the expression pattern is that the mRNA encoding β-galactosidase is
made only when there is a substrate (lactose) present for it to act on and only in the absence of a
better substrate (glucose).
(a) repressed: With succinate as carbon source, an enzyme to break lactose down is not needed
and it would be a waste of energy to produce it. Synthesis of β-galactosidase mRNA is
repressed.
(b) induced: With lactose added to the medium, breaking down lactose to galactose and glucose
will produce a better substrate than succinate, so the synthesis of β-galactosidase mRNA is
induced.
(c) catabolite repressed: When glucose is added to the medium, it is a better substrate than
lactose because it is no longer necessary to break lactose down to produce glucose. The
synthesis of β-galactosidase mRNA is no longer needed and its synthesis is repressed by the
catabolite glucose, hence catalabolite repression.
These effects are all the result of specific effector proteins (a repressor and an activator)
interacting with specific DNA sequences in the control region (operator/promoter) of the DNA of
the lac operon.
z
y
a
p
t o/p
t
i
transcription
mRNA
mRNA
translation
repressor
β-galactosidase
Regulatory "genes"
i encodes the repressor protein
o = operator or repressor binding site
p = promoter
t = terminator
permease transacetylase
Structural genes
z encodes β-galactosidase
y encodes lactose permease
a encodes β-galactoside transacetylase
One additional protein that is required for the control of expression of the lac operon is encoded
by a gene crp that is unlinked to the lac operon (is situated elsewhere on the chromosome).
crp encodes a transcription activator protein variously know as catabolite gene activator
protein (CAP) or cAMP receptor protein (CRP). Either name is acceptable although we will
use CAP in these notes.
8-5
(i) repressed state: growth on succinate
+
o/p
+
CAPcAMP
RNA-P
i
CAP cAMP
The repressor binds to the operator which
overlaps the promoter and prevents RNA
polymerase + CAP from binding.
(RNA-P = RNA polymerase)
active repressor
(can bind to o)
(ii) induced state: lactose added to the growth medium
CAP-cAMP facilitates RNA-P binding to the promoter. In so doing it activates transcription
o/p
i
+
+
RNA-P CAPcAMP
CAP cAMP
mRNA
inactive repressor
(cannot bind to o)
allolactose
Where does the allolactose come from? In fact, the operon is "leaky" and even when repressed
there is a small amount of expression resulting in a small amount of lactose permease and βgalactosidase to bring lactose into the cell and convert it into allolactose. The increase in mRNA
levels can be as much as 1000 times.
Binding of allolactose to the repressor protein causes a change in conformation that prevents
the repressor from binding to the operator. In fact the repressor exhibits allosteric properties and
we can consider it to exist in an equlibrium between the R (active) and T (inactive) states with
allolactose displacing the equilibrium towards the T state.
T
R
8-6
(iii) catabolite repressed state: glucose added to the growth medium (lactose is still present)
glucose causes cAMP levels to
drop reducing the amount of
CAP-cAMP
o/p
i
+
RNA-P CAP
mRNA
(reduced to ~1/3 or
less of fully induced)
inactive repressor
(cannot bind to operator)
allolactose
In fact, the term catabolite repression might actually be considered a misnomer because the
process involves an activator protein (CAP) that becomes less active rather than a repressor
protein. The lac repressor protein remains inactive as well because of the presence of
lactose/allolactose.
The lac operon is regulated by both negative (repression) and positive (activation) mechanisms.
(b) trp operon - negative control in an anabolic (biosynthetic) operon
Lec #24
Tryptophan is produced from intermediates in the pentose phosphate pathway taken through a
very complex series of about 15 reactions. The final six steps are carried out by enzymes
encoded in the trp operon.
The mode of control here is conceptually quite different, in fact the opposite, from that in a
catabolic operon. A catabolic operon is turned ON only when the substrate of the operon is
present in order that energy is not wasted to make mRNA and enzymes that are not needed. An
anabolic operon is turned OFF when the product of the operon enzymes is present, in order that
energy is not wasted to make mRNA and enzymes that are not needed.
trp operon mRNA
glucose
+ tryptophan
induced
repressed
Time
8-7
(i) induced state: growth on glucose
R
RNA-P
o/p
E
D
C
B
A
mRNA
inactive repressor
(cannot bind to operator)
(ii) repressed state: with tryptophan added
R
RNA-P
o/p
E
D
C
B
A
active repressor-corepressor complex
tryptophan
(a corepressor)
3. Example of short and long term control in one pathway
8-8
The following example builds on the trp operon to provide not only an illustration of the three
different types of short term control in a branched pathway but also another example of long term
or transcriptional control. The mode of repressor action in the transcriptional control is exactly
analogous to that in the trp operon in that amino acids act as co-repressors to turn off expression
of certain genes.
You will find the details for this pathway starting on page 6-8.
The control will be broken into two separate stages: short term and long term.
(a) short term: feedback inhibition
aspartate
multiple
aspartate
kinases
I
II
III
aspartyl-P
synergistic
aspartate semialdehyde
multiple
homoserine
II
I
dehydrogenases
homoserine
lysine
homoserine-P
methionine
threonine
α-ketobutyrate
isoleucine
sequential
8-9
(b) long term: transcriptional control
aspartate
I
II
III
aspartyl-P
aspartate semialdehyde
II
I
homoserine
lysine
homoserine-P
methionine
threonine
α-ketobutyrate
isoleucine
Control occurs in two operons:
1. The genes encoding aspartyl kinase I and homoserine dehydrogenase II are part of the
same operon controlled by threonine and isoleucine as corepressors.
2. The genes encoding aspartyl kinase II and homoserine dehydrogenase I are part of
another operon controlled by methionine as a corepressor.
8-10
4. Summary
1. Short term control - feedback inhibition in a linear and branched pathways
2. Long term control - control of transcription
(a) lac operon - repressor (negative control) and CAP-cAMP (poisitive control)
(b) trp operon - repressor and corepressor
3. Example of control mechanisms in the aspartate family of amino acids.
8-11
Integration of Pathways - Overview
UDPG
glc
ribulose-5P
ribose-5P
glc-1P
glycogen
glc-6P
PRPP
CO2
IMP
frc-6P
GMP
AMP
frc-1,6bisP
Ga-3P
ATP
dATP
GTP dGTP
DHA-P
1,3-bisPGA
triglycerides
phospholipids
CTP
UTP
dTTP
thr
2-PGA
ala
val
PEP
UMP
ile
pyruvate
thr
citrulline
OAA
malate
urea
succ
succCoA
NADH
ADP + Pi
ATP
FAD
NAD+
CO2
N2
NH3
glu
CO2
val
FADH2
simple lipids
isocitrate
α-KG
fum
tyr
phe
fatty acids
citrate
glyoxalate
arg-succ
arg
CO2
AcCoA
asp
ornithine
gly
ser
3-PGA
dCTP
NH3
gln