A Eukaryotic Transcriptional Activator Bearing the DNA Specificity of a Prokaryotic Repressor - PowerPoint PPT Presentation

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A Eukaryotic Transcriptional Activator Bearing the DNA Specificity of a Prokaryotic Repressor

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  1. A Eukaryotic Transcriptional Activator Bearing the DNA Specificity of a Prokaryotic Repressor By Roger Brent and Mark Ptashne Cell (1985) 43:729-736 Presented by N. Kuldell and R. Weiss for 20.382 02.10.10

  2. Principles of gene regulation Hypothesize: Tx’n is regulated with modular components Hypothesize: Eukaryotic and prokaryotic systems share common themes for control • Binding • Protein-protein contact to activate • Cooperativity • Modularity Why we care: enable synthetic control systems

  3. Prokaryotic Transcriptional Regulation Repression Activation Lac repressor blocks RNAP figure from Freeman online text http://bcs.whfreeman.com/thelifewire/content/chp13/1302001.html • cI contacts RNAP • figure from The Genetic Switch

  4. Thumbnail sketch about LexA repressor in E. coli SOS response pathway DNA repair gene DNA repair gene UV damage

  5. Thumbnail sketch about GAL regulation in yeast, circa 1985


  6. Modular functions enable synthetic control of transcription Domain swap experiment yeast gene bacterial protein TXN? no Brent and Ptashne Cell (1985) 43:729-736

  7. Modular functions enable synthetic control of transcription Domain swap experiment yeast gene bacterial protein TXN? no yes Yeast activation domain Bacterial binding domain Brent and Ptashne Cell (1985) 43:729-736

  8. “what if”…eukaryotic activators work like l cI • You’re crazy…. • What about nuclear localization signals? • What about histones? Brent Nature (1984) 312:612

  9. “what if”…eukaryotic activators work like l cI • You’re crazy…. • What about nuclear localization signals? • What about histones? So knew that bacterial protein could function in eukaryotic nucleus… Brent Nature (1984) 312:612

  10. “what if”…eukaryotic activators work like l cI • You’re crazy…. • What about nuclear localization signals? • What about histones? • What if it just works differently? Brent Cell (2004) S116:S73

  11. “what if”…eukaryotic activators work like l cI • You’re crazy…. • What about nuclear localization signals? • What about histones? • What if it just works differently? • What about distance between binding site for activator and promoter?

  12. LexA-GAL4 fusion protein construct Brent and Ptashne Cell (1985) 43:729-736

  13. LexA-GAL4 works in E. coli Brent and Ptashne Cell (1985) 43:729-736

  14. LexA-GAL4 activates transcription in yeast

  15. LexA-GAL4 activates transcription in yeast

  16. Mapping 5’ end of transcript to verify Brent and Ptashne Cell (1985) 43:729-736

  17. Squelching by overexpression of GAL4 Brent and Ptashne Cell (1985) 43:729-736

  18. Downstream Activation as well! Figure 5 Brent and Ptashne Cell (1985) 43:729-736

  19. Critique • Key assumptions • protein functions are modular • eukaryotic/prokaryotic/whatever…. • Biggest gaps • footprinting of protein on DNA? • RNAP contact? • nucleosome remodeling? • generalizable?

  20. Significance and Meta-lessons • Protein “parts” can be moved from natural context and intelligently designed to regulate transcription • Activation via binding and contact with RNAP “yeast two hybrid” “bacterial two hybrid”