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Bacteriophage lambda (l)

Bacteriophage lambda (l). Transcriptional switches can regulate cellular decisions. Lysis or Lysogeny. Lysis : Infection by phage produces many progeny and breaks open (lyses) the host bacterium

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Bacteriophage lambda (l)

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  1. Bacteriophage lambda (l) Transcriptional switches can regulate cellular decisions

  2. Lysis or Lysogeny • Lysis: Infection by phage produces many progeny and breaks open (lyses) the host bacterium • Lysogeny: After infection, the phage DNA integrates into the host genome and resides there passively • No progeny • No lysis of the host • Can subsequently lyse (lysogeny) • Bacteriophage lambda can do either.

  3. UV Induction Lysogeny Lysis

  4. + + CIII CII CII gam int red N cI cro cII O P Q S R A…J att xis cIII Pint tL1 PL oL PRM PR tR1 PRE tR2 PR‘ t6S tR3 oR CI Int Lysogeny: CII and CIII stimulate expression of cI to make repressor tint PRE = promoter for repression establishment Repressor

  5. gam int red N cI cro cII O P Q S R A…J att xis cIII Pint tL1 PL oL PRM PR tR1 PRE tR2 PR‘ t6S tR3 oR CI CI CI Lysogeny: Repressor turns off transcription PRM = promoter for repression maintenance Repressor Activated by Repressor binding to oR1 & oR2

  6. l operators overlap promoters oR : oR3 oR2 oR1 PR -35 -10 TTGACT GATAAT cro N TTAGAT 5’ ATAGAT 5’ -10 -35 PRM

  7. Repressor structure l repressor is a dimer; monomer has 236 amino acids. l repressor can bind cooperatively to operator sub-sites.

  8. l-lachybrid genes PlacelcI gene under lac control. Use lacZ as a reporter. lac p, o l cI l pR , OR lacZ 321 Control amount of l repressor by [IPTG]. E. coli with lac repressor, no lacZ. See effect of l repressor by b-galactosidase activity

  9. Repressor stimulates transcription from PRM lac p, o l cI l pRM , OR lacZ 123 b-galactosidase l repressor [IPTG] l repressor at oR1 and oR2 stimulates transcription from pRM.

  10. Binding of repressor blocks transcription from pR but activates pRM PR -35 -10 2 dimers of Repressor, bound cooperatively oR3 RNA Pol cro N oR2 oR1 -10 -35 = operator PRM = promoter -35 -10

  11. Events at initiation of transcription

  12. Let R = RNA polymerase, P = promoter (closed), and Po= promoter (open) ATP + UTP* kf KB RP RPo R + P kr Abortive transcripts ApUp*U [ApUp*U] lag time Abortive initiation assay

  13. 1 1 1 1 1 1 KB kf KB kf [R] [R] kf kf Measure kf and KB from lag time vs. 1/[R] Lag time in abortive initiation assay is inversely proportional to [R]. Lag time = x + Lag time Slope = Y-intercept =

  14. Effect of wild-type and pc mutantλ repressors on activity of PR & PRM

  15. Effect of Operator Mutations on Transcriptional Control of PR&PRM OR1+OR2-OR3+ OR1-OR2+OR3-

  16. Effect of λ-pc mutations on KB and k2

  17. Architecture of λOR

  18. Mutations in the Activating Region ofλ Repressor Glu Glu Glu

  19. Mutations in the δ subunit of RNA polymerase that interfere w/λ repressor-mediated activation of PRM transcription

  20. Effect of mutations in the δ subunit of RNA polymerase on activator-dependent and independent transcription of the lac promoter

  21. A model for interaction of the δ subunit of RNA polymerase with λ repressor

  22. The Awesome Power of Genetics

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