A Eukaryotic Transcriptional Activator Bearing the DNA Specificity of a Prokaryotic Repressor By Roger Brent and Mark Ptashne Cell (1985) 43:729-736 Presented.

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Presentation transcript:

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

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

Prokaryotic Transcriptional Regulation Activation cI contacts RNAP figure from The Genetic Switch Repression Lac repressor blocks RNAP figure from Freeman online text ire/content/chp13/ html

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

Thumbnail sketch about GAL regulation in yeast, circa 1985

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

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

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

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

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

“what if”…eukaryotic activators work like 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?

LexA-GAL4 fusion protein construct Brent and Ptashne Cell (1985) 43:

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

LexA-GAL4 activates transcription in yeast

Mapping 5’ end of transcript to verify Brent and Ptashne Cell (1985) 43:

Squelching by overexpression of GAL4 Brent and Ptashne Cell (1985) 43:

Downstream Activation as well! Brent and Ptashne Cell (1985) 43: Figure 5

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

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” 