Real-time analysis of Oct protein-octamer interaction and transcription complex assembly.
Specific interactions between the protein-binding sequence of the immunoglobulin transcription regulatory element, the octamer, and Oct proteins have been investigated using a biosensor based on surface plasmon resonance. By analysis of in vitro translated Oct1 and Oct2A with a consensus octamer probe, it was shown that the affinity constant, association rate constant and dissociation rate constant of Oct1 were higher than for Oct2A. The biggest difference was in the association rate constants, but this difference was reduced when an octamer motif containing a point mutation was used as a probe. Elements in the octamer flanking 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 approach to study specific protein-DNA and protein-protein interactions in analysis of transcriptional regulation.
Malmborg
A C
AC
Department of Immunotechnology, Lund University, Sweden.
Johansson
K
K
Sigvardsson
M
M
Borrebaeck
C A
CA
Leanderson
T
T
D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D015374:Biosensing Techniques; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D051863:Host Cell Factor C1; D006801:Humans; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D050811:Octamer Transcription Factor-1; D050813:Octamer Transcription Factor-2; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D013698:Templates, Genetic; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000276:immunology
D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D018398:Homeodomain Proteins; D051863:Host Cell Factor C1; D007145:Immunoglobulin kappa-Chains; D050811:Octamer Transcription Factor-1; D050813:Octamer Transcription Factor-2; C494165:POU2F1 protein, human; C058435:POU2F2 protein, human; D010455:Peptides; D014157:Transcription Factors