REAL-TIME ANALYSIS OF OCT PROTEIN-OCTAMER INTERACTION AND TRANSCRIPTION COMPLEX ASSEMBLY

Specific interactions between the protein-binding sequence of the immu noglobulin transcription regulatory element, the octamer, and Oct prot eins have been investigated using a biosensor based on surface plasmon resonance. By analysis of in vitro translated Oct1 and Oct2A with a c onsensus octamer probe, it was shown that the affinity constant, assoc iation rate constant and dissociation rate constant of Oct1 were highe r than for Oct2A. The biggest difference was in the association rate c onstants, but this difference was reduced when an octamer motif contai ning a point mutation was used as a probe. Elements in the octamer fla nking sequence could increase the on-rate of Oct proteins to a mutated octamer while not decreasing the off-rate. Oct-octamer interaction in whole nuclear extracts could be detected readily in the biosensor and adapter interactions with template bound proteins were revealed. Thus , biosensor analysis represent a fast and convenient alternative appro ach to study specific protein-DNA and protein-protein interactions in analysis of transcriptional regulation.