distinguish between positive and negative transcriptional regulation make predictions based on...

• 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

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

lactose

galactose

glycerol

maltose

sorbose

Sugar Utilized

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

The arabinose regulon

Figure 12.18

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

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

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

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

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

Is araC a Positive or Negative Regulator?

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

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

Negative Regulation & Constitutive Mutants

No inducer - Gene will be ON/OFF?

Gene YPromoter

Negative Regulation & Constitutive Mutants

Plus inducer - Gene will be ON/OFF?

Gene YPromoter

• 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

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

Positive Regulation & Constitutive Mutants

No inducer - Gene will be ON/OFF?

Gene YPromoter

Positive Regulation & Constitutive Mutants

Plus inducer - Gene will be ON/OFF?

Gene YPromoter

• 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

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

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’

• 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

Gene YPromoter

Wild type

No L-ara

OFFAraC

Wild Type Plus L-ara

Gene YPromoter

RNAPol

ONAraC

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’

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

Fig 12.20