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Biochemical analysis of the Kruppel-associated box (KRAB) transcriptional repression domain.

The Kruppel-associated box (KRAB) domain is a 75-amino acid transcriptional repressor module commonly found in eukaryotic zinc finger proteins. KRAB-mediated gene silencing requires binding to the RING-B box-coiled-coil domain of the corepressor KAP-1. Little is known about the biochemical properties of the KRAB domain or the KRAB.KAP-1 complex. Using purified components, a combination of biochemical and biophysical analyses has revealed that the KRAB domain from the KOX1 protein is predominantly a monomer and that the KAP-1 protein is predominantly a trimer in solution. The analyses of electrophoretic mobility shift assays, GST association assays, and plasmon resonance interaction data have indicated that the KRAB binding to KAP-1 is direct, highly specific, and high affinity. The optical biosensor data for the complex was fitted to a model of a one-binding step interaction with fast association and slow dissociation rates, with a calculated K(d) of 142 nm. The fitted R(max) indicated three molecules of KAP-1 binding to one molecule of the KRAB domain, a stoichiometry that is consistent with quantitative SDS-polyacrylamide gel electrophoresis analysis of the complex. These structural and dynamic parameters of the KRAB/KAP-1 interaction have implications for identifying downstream effectors of KAP-1 silencing and the de novo design of new repression domains.

Pubmed ID: 10748030

Authors

  • Peng H
  • Begg GE
  • Harper SL
  • Friedman JR
  • Speicher DW
  • Rauscher FJ

Journal

The Journal of biological chemistry

Publication Data

June 16, 2000

Associated Grants

None

Mesh Terms

  • Amino Acid Sequence
  • Binding Sites
  • Circular Dichroism
  • DNA-Binding Proteins
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli
  • Molecular Sequence Data
  • Molecular Weight
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Secondary
  • Recombinant Proteins
  • Repressor Proteins
  • Structure-Activity Relationship
  • Zinc Fingers