Transcription factor E2F binds DNA as a heterodimer.
E2F is a mammalian transcription factor that appears to play an important role in cell cycle control. DNA affinity column-purified E2F from HeLa cells reproducibly exhibits multiple protein bands when analyzed by SDS/PAGE. After electrophoretic purification, electroelution, and refolding of the individual protein components, the E2F DNA binding activity of the individual proteins was poor. However, upon mixing the individual components together, a dramatic (100- to 1000-fold) increase in specific DNA binding activity was observed. The five protein bands isolated can be separated into two groups based on apparent molecular mass. Optimal reconstitution of activity requires one of the two proteins found in the group of larger molecular mass (approximately 60 kDa) and one of the three proteins in the smaller-sized group (approximately 50 kDa). The reconstituted heterodimer is identical to authentic affinity-purified E2F by three criteria: DNA-binding specificity, DNA pattern, and binding to the retinoblastoma gene product. A recently cloned protein with E2F-like activity, RBP3/E2F-1, is related to the protein components of the group of larger molecular mass, as determined by Western blot analysis and reconstitution experiments. These data suggest that E2F, like many other transcription factors, binds DNA as an oligomeric complex composed of at least two distinct proteins.
Huber
H E
HE
Department of Cancer Research, Merck Research Laboratories, West Point, PA 19486.
Edwards
G
G
Goodhart
P J
PJ
Patrick
D R
DR
Huang
P S
PS
Ivey-Hoyle
M
M
Barnett
S F
SF
Oliff
A
A
Heimbrook
D C
DC
D000260:Adenoviruses, Human / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D050684:E2F Transcription Factors; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D056465:Retinoblastoma-Binding Protein 1; D013379:Substrate Specificity; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D004247:DNA; D005982:Glutathione Transferase