a eukaryotic transcriptional activator bearing the dna specificity of a prokaryotic repressor
DESCRIPTION
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. Principles of gene regulation. - PowerPoint PPT PresentationTRANSCRIPT
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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
![Page 2: A Eukaryotic Transcriptional Activator Bearing the DNA Specificity of a Prokaryotic Repressor](https://reader036.vdocuments.site/reader036/viewer/2022062314/5681438a550346895db00905/html5/thumbnails/2.jpg)
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
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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
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Thumbnail sketch about LexA repressor in E. coli
DNA repair gene
SOS response pathway
UV damage
DNA repair gene
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Thumbnail sketch about GAL regulation in yeast, circa 1985
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Modular functions enable synthetic control of transcription
Domain swap experiment
bacterial protein
Brent and Ptashne Cell (1985) 43:729-736
yeast gene
TXN?no
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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
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“what if”…eukaryotic activators work like cI
You’re crazy….•What about nuclear localization signals?•What about histones?
Brent Nature (1984) 312:612
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“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…
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“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
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“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?
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LexA-GAL4 fusion protein construct
Brent and Ptashne Cell (1985) 43:729-736
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LexA-GAL4 works in E. coli
Brent and Ptashne Cell (1985) 43:729-736
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LexA-GAL4 activates transcription in yeast
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LexA-GAL4 activates transcription in yeast
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Mapping 5’ end of transcript to verify
Brent and Ptashne Cell (1985) 43:729-736
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Squelching by overexpression of GAL4
Brent and Ptashne Cell (1985) 43:729-736
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Downstream Activation as well!
Brent and Ptashne Cell (1985) 43:729-736
Figure 5
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CritiqueKey assumptions
•protein functions are modular•eukaryotic/prokaryotic/whatever….
Biggest gaps
•footprinting of protein on DNA? •RNAP contact?•nucleosome remodeling? •generalizable?
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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”