Transcript lectureFeb27

Transcriptional Regulation of the L-ara
operon
• Distinguish between positive and negative
transcriptional regulation
• Make predictions based on hypotheses
• Infer protein function from mutant phenotype
Pages 519-525 of textbook
Originally written by Lazar Dimitrov
Example of catabolite control
1.Grow cells in minimal media with lactose as the
carbon course
2. Add Glucose
3. Measure Beta-galactosidase-LacZ
-Cell default setting is to use glucose for energy
production
Sugar Utilized
lactose
galactose
glycerol
maltose
sorbose
Regulation of cAMP Production
PEP (phospho enol pyruvate)-dependent sugar
phosphotransferase system-transports glucose
into the cells
-PTS IIAglc exists in two form +/- phosphate
-phospho IIAglc activates adenylate cyclase
-ration of IIAglc to IIAglc-P depends on glucose
availability
-Hpr Histidine protein adds phosphates
Glucose
Glycolysis
PEP:Pyruvate
TCA Cycle
The arabinose regulon
Figure 12.18
Mutant Analysis
Lactose
Galactose &
Glucose
lacY,
lacZ
Negative Regulation (lacI)
Glycolysi
s
L-arabinose Utilization
Larabinose
L-ribulose
L-ribulose5phosphate
Is it subject to positive or negative
regulation?
D-xylulose5phosphate
How Many Genes?
Replica
plate
Mutagenesi
s
Minimal
Glucose
Minimal
Larabinose
Ara- mutants
• Test each Ara- for dominance/recessivity to w.t.
• Set up complementation tests between all possible
recessive Ara- mutant pairs
How Many Genes?
• At least 4 complementation
groups/genes (araA, araB, araC and
araD) are defined by the Ara- mutants
Larabinose
araA
L-ribulose
•What about araC
araB
L-ribulose5phosphate
araD
D-xylulose5phosphate
araC- Mutants Are “Supersuppressed”
OFF
C-
BAD
No L-arabinose
OFF
C-
BAD
Plus Larabinose
• What could be the function of araC?
(Hint: araC is not the L-ara permease)
Is araC a Positive or Negative
Regulator?
C-
OFF
BAD
No Larabinose
I-
ON
ZYA
No Lactose
C-
OFF
BAD
Plus L-arabinose
I-
ON
ZYA
Plus
Lactose
Is araC a Positive or Negative Regulator?
• What mutations in the lac operon cause “supersuppressed” phenotype?
Negative Regulation
Wild type
No
inducer
Promoter
Wild Type
Plus
inducer
OFF
RNAPol
ON
Gene Y
Promoter
Gene Y
Constitutive Mutants
Loss-of-function
(LOF) mutations
RNAPol
ON
Promoter
Gene Y
Negative Regulation &
Constitutive Mutants
Promoter
Gene Y
No inducer - Gene will be ON/OFF?
Negative Regulation &
Constitutive Mutants
Promoter
Gene Y
Plus inducer - Gene will be ON/OFF?
Negative Regulation &
Constitutive Mutants
• Partial diploid containing a constitutive
allele and a wild type allele has wild type
phenotype
• THEREFORE, constitutive mutants are
dominant/recessive to wild type under
negative regulation
Positive Regulation
Wild type
No
inducer
Wild Type
Plus
inducer
RNAPol
ON
OFF
Promoter
Gene Y
Promoter
Constitutive Mutants
RNAPol
Gain-of-function
(GOF) mutations
ON
Promoter
Gene Y
Gene Y
Positive Regulation &
Constitutive Mutants
Promoter
Gene Y
No inducer - Gene will be ON/OFF?
Positive Regulation &
Constitutive Mutants
Promoter
Gene Y
Plus inducer - Gene will be ON/OFF?
Positive Regulation &
Constitutive Mutants
• Partial diploid containing a constitutive allele
and a wild type allele has a constitutive
phenotype
• THEREFORE, constitutive mutants are
dominant/recessive to wild type under
positive regulation
Hypothesis
• araC is an activator of the L-ara operon
araCNo L-ara
araCPlus L-ara
OFF
Promoter
Gene Y
OFF
Promoter
Gene Y
Predictions
•araCc will be dominant/recessive to wild type araC
•Constitutive mutants, i.e. araCc, will be
rare/common?
•araC- mutants cannot be induced with L-ara
Bahavior of araCc mutants
C+ BA D
F’
C+ BA D
F’
Cc
OFF
BAD
No L-arabinose
ON
Cc
BAD
Plus Larabinose
Conclusion: araCc mutations are
dominant/recessive to wild type araC
• If araCc mutations are recessive to wild
type, is araC a repressor?
• If araC is a repressor, why are araCc
mutations supersuppressive and not
constitutive?
• If araC is a repressor, why are araCc
mutations rare?
AraC a repressor & an activator
• Revised Hypothesis: AraC is a repressor
in the absence of L-ara but an activator in
the presence of L-ara
Wild type
No L-ara
Wild Type
Plus L-ara
RNAPol
AraC
Promoter
OFF
Gene Y
AraC
Promoter
ON
Gene Y
AraC a repressor & an activator
• BUT … why is araCc recessive to wild
type?
C+ BA D
F’
AraCc
AraC
AraCc
OFF
Cc
AraC
AraCc
AraCc
AraC
AraCc
BAD
AraC
AraC
No Larabinose
AraC a repressor & an activator
• At least two models (not mutually exclusive)
– AraC in its repressor conformation binds to DNA with
higher affinity than AraCc mutants
– AraC peptides form a complex (e.g. a homodimer) to
be functional
RNAPol
AraC AraC
Promoter
OFF
Gene Y
AraC
AraC
Promoter
ON
Gene Y
Fig 12.20