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Transcriptional Regulation in the L-ara Operon: Predictions and Mutant Phenotype Analysis

Understand positive and negative transcriptional regulation in the L-ara operon, predict outcomes based on hypotheses, and infer protein function from mutant phenotype analysis. Explore the catabolite control mechanism and gene mutants affecting sugar utilization. Discover the roles of araC and araCc mutations in the operon regulation.

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Transcriptional Regulation in the L-ara Operon: Predictions and Mutant Phenotype Analysis

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  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 PEP:Pyruvate 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. Ara- mutants 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 typeNo inducer Wild Type Plus inducer ON OFF RNAPol Promoter Gene Y Promoter Gene Y Constitutive Mutants Loss-of-function (LOF) mutations ON RNAPol Promoter Gene Y

  14. Negative Regulation & Constitutive Mutants Promoter Gene Y No inducer - Gene will be ON/OFF?

  15. Negative Regulation & Constitutive Mutants Promoter Gene Y 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 Promoter Gene Y Promoter Gene Y Constitutive Mutants RNAPol Gain-of-function (GOF) mutations ON Promoter Gene Y

  18. Positive Regulation & Constitutive Mutants Promoter Gene Y No inducer - Gene will be ON/OFF?

  19. Positive Regulation & Constitutive Mutants Promoter Gene Y 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 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

  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 Promoter Gene Y Promoter Gene Y

  25. AraCc AraCc AraCc AraCc AraCc AraC AraC AraC AraC AraC 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 Promoter Gene Y Promoter Gene Y

  27. Fig 12.20

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