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 Galactose & Glycolysi Lactose lacY, Glucose s lacZ Negative Regulation (lacI) L-arabinose Utilization L- L-ribulose- D-xylulose- L-ribulose 5- 5- arabinose phosphate phosphate Is it subject to positive or negative regulation? How Many Genes? Replica plate Minimal Mutagenesi Minimal L- s Glucose arabinose 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- araA araB L-ribulose- araD D-xylulose- L-ribulose 5- 5- arabinose phosphate phosphate •What about araC araC- Mutants Are “Super- suppressed” OFF OFF C- BAD C- BAD Plus L- No L-arabinose arabinose • What could be the function of araC? (Hint: araC is not the L-ara permease) Is araC a Positive or Negative Regulator? OFF OFF C- BAD C- BAD No L- Plus L-arabinose arabinose ON ON I- ZYA I- ZYA Plus No Lactose Lactose Is araC a Positive or Negative Regulator? • What mutations in the lac operon cause “super- suppressed” phenotype? Negative Regulation Wild type Wild Type No Plus inducer inducer OFF RNAPol ON Promoter 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 Wild Type No Plus inducer inducer RNAPol OFF ON Promoter Gene Y Promoter Gene Y Constitutive Mutants RNAPol Gain-of-function (GOF) mutations ON Promoter 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 araC- - araC Plus L-ara No L-ara OFF OFF Promoter Gene Y 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 C+ BA D F’ F’ OFF ON Cc BAD Cc BAD Plus L- No L-arabinose arabinose 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 Wild Type No L-ara Plus L-ara RNAPol AraC OFF AraC ON Promoter Gene Y Promoter Gene Y AraC a repressor & an activator • BUT … why is araCc recessive to wild type? C+ BA D AraCc ’ F AraC AraC AraCc AraCc OFF AraCc AraC AraCc Cc BAD AraC AraC No L- arabinose 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 OFF AraC AraC ON Promoter Gene Y Promoter Gene Y Fig 12.20.