Single Order Binding Reaction

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Single Order Binding Reaction
KD
___PS___ Sfree PS
fractional occupancy
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Equilibrium dissociation constant
association
dissociation
P free DNA binding protein S free DNA
binding sites
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Operational Definition for Kd The concentration
of DNA binding protein when half the sites are
occupied at equilibrium
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Electromobility Shift Assay
DNA at ltltKD
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Yeast Nucleus 1500 nm in diameter
Yeast Genome 1.2 X 107 bp
DNA concentration is WAY above the KD for both
specific and non-specific binding
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Rick Youngs Genomic Binding site experiment
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IP-enriched DNA
Un-enriched DNA
Merge
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Conventional Chromatin Immunoprecipitation Assay
Gal4
Gal4
Gal1
UAS
UAS
Gal4
Gal3
UAS
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Gal4
Gal4
Gal4
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New Observations 1. Three new target genes of
Gal4 2. Ten binding sites in entire genome! gt
200 Gal4 sites are encoded in the yeast
genome KD constant predicts even more Gal4
bound 3. Some target genes bound by Gal4 even
though they are not expressed
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• Observations
• Many transcription factor binding sites exist in
  the genome, but only a small fraction are
  occupied why? How?
• Many silent/repressed target genes are occupied
  by their transcription activator. why? How?

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353 commonly regulated genes?
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Regulatory Circuits
Feed Forward Circuit
Autoregulatory circuit
Core Circuit?
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• Summary
• Transcription Factors are bound to fewer sites
  than exist in the genome preference for
  intergenic sites in promoters/enhancers
• Transcription Factors may be bound to silent
  targets (gene expression arrays compared with
  ChiP/ChIP arrays) - a need for negative
  regulators
• Different classes of Transcription Factors
  cooperate to regulate gene targets circuitry