distinguish between positive and negative transcriptional regulation make predictions based on...
TRANSCRIPT
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• Distinguish between positive and negative transcriptional regulation
• Make predictions based on hypotheses• Infer protein function from mutant
phenotypePages 519-525 of textbook
Transcriptional Regulation of the L-ara operon
Originally written by Lazar Dimitrov
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Example of catabolite control
1.Grow cells in minimal media with lactose as the carbon course2. Add Glucose3. Measure Beta-galactosidase-LacZ-Cell default setting is to use glucose for energy production
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lactose
galactose
glycerol
maltose
sorbose
Sugar Utilized
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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
Regulation of cAMP Production
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The arabinose regulon
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Figure 12.18
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Mutant Analysis
Lactose
Galactose & GlucoselacY,
lacZ
Glycolysis
Negative Regulation (lacI)
Is it subject to positive or negative regulation?
L-arabinos
e
L-ribulose
L-ribulose-
5-phosphat
e
D-xylulose-
5-phosphat
e
L-arabinose Utilization
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How Many Genes?
Mutagenesis
Minimal Glucose
Minimal L-
arabinose
Replica plate
Ara- mutants
• Test each Ara- for dominance/recessivity to w.t.• Set up complementation tests between all
possible recessive Ara- mutant pairs
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How Many Genes?
• At least 4 complementation groups/genes (araA, araB, araC and araD) are defined by the Ara- mutants
L-arabinos
e
L-ribulose
L-ribulose-
5-phosphat
e
D-xylulose-
5-phosphat
e
araA araB araD
•What about araC
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araC- Mutants Are “Super-suppressed”
C- B A D
OFF OFF
No L-arabinose
Plus L-arabinose
• What could be the function of araC? (Hint: araC is not the L-ara permease)
C- B A D
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C- B A DOFF
C- B A DOFF
No L-arabinose
Plus L-arabinose
Is araC a Positive or Negative Regulator?
I- Z Y AON
No Lactose
I- Z Y AON
Plus Lactose
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Is araC a Positive or Negative Regulator?
• What mutations in the lac operon cause “super-suppressed” phenotype?
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Negative RegulationWild Type
Plus inducer
Gene YPromoter
RNAPol ONGene YPromoter
Wild typeNo
inducer
OFF
Constitutive Mutants
Gene YPromoter
RNAPol ON
Loss-of-function(LOF) mutations
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Negative Regulation & Constitutive Mutants
No inducer - Gene will be ON/OFF?
Gene YPromoter
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Negative Regulation & Constitutive Mutants
Plus inducer - Gene will be ON/OFF?
Gene YPromoter
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• 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
Negative Regulation & Constitutive Mutants
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Positive Regulation
Gene YPromoter
Wild typeNo
inducer
Wild Type Plus
inducer
Gene YPromoter
RNAPol
ONOFF
Constitutive Mutants
Gene YPromoter
RNAPol
ONGain-of-function(GOF) mutations
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Positive Regulation & Constitutive Mutants
No inducer - Gene will be ON/OFF?
Gene YPromoter
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Positive Regulation & Constitutive Mutants
Plus inducer - Gene will be ON/OFF?
Gene YPromoter
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• 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
Positive Regulation & Constitutive Mutants
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Hypothesis• araC is an activator of the L-ara operon
Gene YPromoter
araC-
No L-ara
OFFGene YPromoter
araC-
Plus L-ara
OFF
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
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Bahavior of araCc mutants
Conclusion: araCc mutations are dominant/recessive to wild type araC
Cc B A DOFF
Cc B A DON
No L-arabinose
Plus L-arabinose
C+ BAD
F’
C+ BAD
F’
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• 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?
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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
Gene YPromoter
Wild type
No L-ara
OFFAraC
Wild Type Plus L-ara
Gene YPromoter
RNAPol
ONAraC
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AraC a repressor & an activator
• BUT … why is araCc recessive to wild type?
Cc B A D
OFF
No L-arabinose
AraC
AraC
AraC
AraCAraC
AraCc
AraCc
AraCc
AraCc
AraCc
C+ BAD
F’
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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
Gene YPromoter
OFFAraCAraCGene YPromoter
RNAPol
ONAraCAraC
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Fig 12.20