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Distinguish between positive and negative transcriptional regulation Make predictions based on hypotheses Infer protein

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.

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Distinguish between positive and negative transcriptional regulation Make predictions based on hypotheses Infer protein

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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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