Transcript Slides PPT

Transcription Regulation
The Lac Operon – a paradigm for
all transcription regulation
Background:
• E. coli (we are back with these pesky little
coliforms) have the ability to grow in media
which contain lactose as their sole carbon
source. Lactose is a sugar found in milk (a
disaccharide).
• To metabolise this sugar the bugs must produce
two enzymes, beta galactosidase and lac
permease.
• If the E. coli are grown up on media with other
carbon sources there is very little activity of
these two enzymes.
Lactose
CH 2OH
CH 2OH
O
OH
O
O
OH
H
reducing end
OH
H
OH
OH
OH
glucose
galactose
lactose
Background:
• The lac permease allows the lactose to
enter the cell.
• The beta galactosidase cleaves the bond
joining the two monosaccharides (known
as a beta galactoside bond, a type of
glycosidic bond).
• Once the sugar has been cleaved the two
monosaccharides can be utilized by the
cell’s glycolytic “house keeping” enzymes.
Is this increase in enzyme
activity a transcriptional event or
simply an activation/deactivation
of pre-existing enzyme activity?
• The answer is it is a transcriptional event.
To test for this a protein synthesis inhibitor
is included in the incubation. The induction
does not occur. (You will show this in the
lab experiment).
How does the presence of
lactose, as the sole carbon
source, control the level of
transcription of the enzymes
that catalyse its utilization?
Two kinds of factors
• Jacob and Monod were able to identify 2
different acting factors; cis and trans
acting factors.
• Cis acting factors only control transcription
on the same piece of DNA
• Trans acting factors influence transcription
on other pieces of DNA
The Lac Operon
• They proposed a model containing the
following elements; lac I, lac Z, Y and A.
• The gene product of Lac I is a protein
repressor, which binds to a region on the
DNA known as the operator (-10 – 0)
• There are only a few copies of the
repressor in the cell and it binds as a
tetramer.
The Lac Operon
• When the repressor is bound to the operator
region it masks the -10 region of the promoter
and prevents the binding of RNA polymerase to
the promoter  NO transcription.
• The actual structural genes are lac Z (beta
galactosidase), Y (lac permease) and A (lac
acetylase).
• These three genes are all transcribed in one
long mRNA, known as a polycistronic mRNA.
How does it work?
• The lac operon is under two forms of
control; positive and negative control.
• Negative control occurs when the binding
of a protein prevents an event. The brake
on the car
• Positive control is when the binding
causes the event. The accelerator on the
car.
Negative control
• The lac repressor (product of gene lac I)
is negative control.
• When lactose is absent the repressor is
bound to the operator preventing RNA
polymerase binding  NO transcription.
• End Result: when there is no substrate
you don’t make the enzyme.
What happens when lactose
enters the cell?
• A small amount of it is converted to
allolactose (this is NOT a substrate for
beta galactosidase)
• The allolactose then binds to the repressor
• The repressor: allolactose complex
dissociates from the operator
• Transcription can begin.
Positive Control
• This is exerted by a protein known as the
Catabolite Activator Protein (CAP)
• This protein binds to a site on the DNA
within the promoter region and increases
the rate of RNA polymerase binding;
hence transcription initiation.
• It only does this when complexed to cAMP.
What is cAMP?
• This is a compound synthesised from ATP
by adenylyl cyclase which acts in both
eukaryotes and prokaryotes as a second
messenger.
• In this situation cAMP ↓ when glucose
enters the bacterial cell
• This causes the cAMP associated with
CAP to come off  CAP without cAMP
then comes off the DNA
Putting it all together
• When lactose is present the
repressor is off the operator.
• When glucose is present the
CAP is off the promoter.
Different Scenarios
1.
2.
3.
4.
Lactose (+) and glucose (-)
Lactose (+) and glucose (+)
Lactose (-) and glucose (+)
Lactose (-) and glucose (-)
Scenario 1
Lactose (+) glucose (-)
•
Some of the Lactose entering the cell
via the few lac permease transporters
available has been converted to
allolactose and has resulted in the
removal of the repressor from the
operator.
Scenario 1
Lactose (+) glucose (-)
•
•
The promoter is now unmasked and
RNA polymerase can now bind and
initiate transcription.
However it won’t do this very
frequently without the help of the
cAMP-CAP bound to the activation
site.
Scenario 1
•
•
This protein complex binding puts a 90o
kink in the DNA and interacts with the
alpha subunit of RNA polymerase.
Without the cAMP:CAP the lac promoter
is a weak promoter varying significantly
from the consensus sequence at -10 and
-35. The combination of the two controls
means beta gal and lac permease are
transcribed at high levels.
Scenario 1
•
•
•
•
Lactose (+) glucose (-)
Repressor with allolactose bound is now off
the operator and the CAP with cAMP bound is
on the DNA in a region upstream from the
promoter.
The RNA pol can bind to the promoter and the
CAP:cAMP can act positively to enhance the
frequency of transcription initiation.
Brake off accelerator on
Scenario 2
Lactose (+) and glucose (+)
• The repressor is off the operator but the
CAP protein (without cAMP) is not bound
to the DNA so initiation only occurs at a
low rate  little transcription.
• Brake off but accelerator off also
Scenario 3
Lactose (-) and glucose (+)
• The repressor is bound to the operator
and the CAP (without cAMP) is not
bound to the DNA. Very little transcription
of the lac operon genes is happening
now.
• Brake on and accelerator off…not going
anywhere fast
Scenario 4
Lactose (-) and glucose (-)
• The cell is starving! The repressor is on
the operator but the cAMP CAP is on the
DNA. If the repressor is bound there is
no transcription  RNA polymerase has
no access.
• Brake on and accelerator on (but you
have run out of petrol!)
Terms
•
•
•
Negative and Positive Control
Cis and Trans acting factors
Global and Specific regulation.
Global Regulation
•
•
A factor can influence the transcription of
a number of genes scattered throughout
the genome e.g. CAP.
A number of genes which encode
catabolic enzymes involved in
carbohydrate metabolism are controlled
by CAP e.g. arabinose operon
Specific Regulation
•
•
A factor can influence the transcription of
a few genes downstream e.g. lac
repressor
Only the next string of genes in the
operon, which are transcribed to a single
polycistronic meassenger RNA will be
affected.
Other Operons
•
•
•
The most famous is the Trp operon.
The 5 enzymes which synthesise
tryptophan are encoded on genes TrpE –
TrpA.
These genes are switched OFF when
there is high [trp] in the cell and switched
ON when [trp] is low.
Trp Operon
•
•
•
•
The opposite to the Lac Operon.
Because the enzymes are anabolic.
The trp repressor binds to the operator
DNA when the trp is bound.
When the trp dissociates from the
repressor i.e. when intracellular trp falls,
the repressor comes off the operator and
transcription can begin.