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

By Roger Brent and Mark PtashneCell (1985) 43:729-736

Presented by N. Kuldell and R. Weiss for 20.38202.10.10

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

http://bcs.whfreeman.com/thelifewire/content/chp13/1302001.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) 43:729-736

yeast gene

TXN?no

Modular functions enable synthetic control of transcription

Domain swap experiment

bacterial protein

Brent and Ptashne Cell (1985) 43:729-736

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) 43:729-736

LexA-GAL4 works in E. coli

Brent and Ptashne Cell (1985) 43:729-736

LexA-GAL4 activates transcription in yeast

LexA-GAL4 activates transcription in yeast

Mapping 5’ end of transcript to verify

Brent and Ptashne Cell (1985) 43:729-736

Squelching by overexpression of GAL4

Brent and Ptashne Cell (1985) 43:729-736

Downstream Activation as well!

Brent and Ptashne Cell (1985) 43:729-736

Figure 5

CritiqueKey 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”