Format

Send to

Choose Destination
J Biol Chem. 2000 Jun 16;275(24):18000-10.

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

Author information

1
Wistar Institute, Philadelphia, Pennsylvania 19104, USA.

Abstract

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.

PMID:
10748030
DOI:
10.1074/jbc.M001499200
[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for HighWire
Loading ...
Support Center