Expression of the Oct-1 transcription factor and characterization of its interactions with the Bob1 coactivator.
The Oct-1 transcription factor regulates a variety of tissue-specific and general housekeeping genes by recruiting specialized coactivators of transcription. It acts synergistically with the B-cell-specific coactivator Bob1 (OCA-B, OBF-1) to stimulate transcription of immunoglobulin genes. To analyze Oct-1's interactions with Bob1 and other regulatory proteins, we have overexpressed and purified different functional domains of the recombinant proteins. A version of Oct-1 that encompasses the amino-terminal activation region and the POU DNA-binding domain was extensively characterized (OctDeltaC1; comprising residues 1-445). Using an in vitro transcription assay, we demonstrate that this fragment is sufficient and necessary to stimulate transcription from an immunoglobulin promoter with Bob1. It also coactivates from the herpes simplex virus ICPO promoter element in the presence of VP16. Using a range of spectroscopic and biophysical techniques, we demonstrate that the activation domains of Oct-1 and Bob1 have little globular structure and that they do not physically interact. Thus, their functional synergy is likely to arise by the co-recruitment of common factors as part of a larger regulatory assembly. We propose a hypothesis to explain why the activation domains of these and other transcription factors of metazoans have little if any intrinsic structure.
Lee
L
L
Department of Biochemistry, Cambridge University, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
Stollar
E
E
Chang
J
J
Grossmann
J G
JG
O'Brien
R
R
Ladbury
J
J
Carpenter
B
B
Roberts
S
S
Luisi
B
B
D000595:Amino Acid Sequence; D002151:Calorimetry; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004357:Drug Synergism; D005822:Genetic Vectors / Q000378:metabolism; D051863:Host Cell Factor C1; D006801:Humans; D008969:Molecular Sequence Data; D050811:Octamer Transcription Factor-1; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012542:Scattering, Radiation; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics; D014965:X-Rays
D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; D009838:Oligodeoxyribonucleotides; C091546:POU2AF1 protein, human; C494165:POU2F1 protein, human; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA