Distinguish between positive and negative transcriptional regulation

1
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
2
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
3
Sugar Utilized
lactose
galactose
glycerol
maltose
sorbose
4
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 PEPPyruvate TCA Cycle
5
The arabinose regulon
6
Figure 12.18
7
Mutant Analysis
Galactose Glucose
Glycolysis
Lactose
lacY, lacZ
Negative Regulation (lacI)
L-arabinose Utilization
L-arabinose
L-ribulose
L-ribulose-5-phosphate
D-xylulose-5-phosphate
Is it subject to positive or negative
regulation?
8
How Many Genes?
Replica plate
Mutagenesis
Minimal Glucose
Minimal L-arabinose

• Test each Ara- for dominance/recessivity to w.t.
• Set up complementation tests between all possible
  recessive Ara- mutant pairs

9
How Many Genes?

• At least 4 complementation groups/genes (araA,
  araB, araC and araD) are defined by the Ara-
  mutants

araA
araB
araD
L-arabinose
L-ribulose
L-ribulose-5-phosphate
D-xylulose-5-phosphate

• What about araC

10
araC- Mutants Are Super-suppressed
OFF
OFF
C-
B
A
D
C-
B
A
D
No L-arabinose
Plus L-arabinose

• What could be the function of araC? (Hint araC
  is not the L-ara permease)

11
Is araC a Positive or Negative Regulator?
OFF
OFF
C-
B
A
D
C-
B
A
D
No L-arabinose
Plus L-arabinose
ON
ON
I-
Z
Y
A
I-
Z
Y
A
No Lactose
Plus Lactose
12
Is araC a Positive or Negative Regulator?

• What mutations in the lac operon cause
  super-suppressed phenotype?

13
Negative Regulation
Wild Type Plus inducer
Wild typeNo inducer
ON
OFF
RNAPol
Gene Y
Promoter
Gene Y
Promoter
Constitutive Mutants
Loss-of-function (LOF) mutations
ON
RNAPol
Gene Y
Promoter
14
Negative Regulation Constitutive Mutants
Gene Y
Promoter
No inducer - Gene will be ON/OFF?
15
Negative Regulation Constitutive Mutants
Gene Y
Promoter
Plus inducer - Gene will be ON/OFF?
16
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

17
Positive Regulation
Wild typeNo inducer
Wild Type Plus inducer
RNAPol
ON
OFF
Gene Y
Promoter
Gene Y
Promoter
Constitutive Mutants
RNAPol
Gain-of-function (GOF) mutations
ON
Gene Y
Promoter
18
Positive Regulation Constitutive Mutants
Gene Y
Promoter
No inducer - Gene will be ON/OFF?
19
Positive Regulation Constitutive Mutants
Gene Y
Promoter
Plus inducer - Gene will be ON/OFF?
20
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

21

• Hypothesis
• araC is an activator of the L-ara operon

araC-Plus L-ara
araC-No L-ara
OFF
OFF
Gene Y
Promoter
Gene Y
Promoter

• 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

22
Bahavior of araCc mutants
B
A
B
A
D
D
C
C
F
F
OFF
ON
Cc
B
A
D
Cc
B
A
D
No L-arabinose
Plus L-arabinose
Conclusion araCc mutations are
dominant/recessive to wild type araC
23

• 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?

24
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 typeNo L-ara
Wild Type Plus L-ara
RNAPol
ON
OFF
AraC
AraC
Gene Y
Promoter
Gene Y
Promoter
25
AraC a repressor an activator

• BUT why is araCc recessive to wild type?

B
A
D
C
F
OFF
Cc
B
A
D
No L-arabinose
26
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
ON
OFF
AraC
AraC
AraC
AraC
Gene Y
Promoter
Gene Y
Promoter
27
Fig 12.20