Retinoblastoma protein reverses DNA bending by transcription factor E2F.
E2F is a mammalian transcription factor involved in cell cycle regulation. The retinoblastoma gene product, pRB, binds to E2F in a cell cycle-dependent manner and appears to turn E2F from a transcriptional activator into a repressor. We show here that in vitro binding of pRB has three major effects on the DNA binding properties of E2F affinity-purified from HeLa cells; pRB binding increases the half-life of E2F.DNA complexes 10-15-fold, it reduces E2F specific DNA binding in the presence of nonspecific DNA by sequestering E2F, and it partially reverses the DNA bending induced by E2F. Upon specific DNA binding, E2F induces a DNA bend with a flexure angle of 125 degrees. Both full-length pRB105 and the N-terminally truncated pRB60 bind to the E2F.DNA complex with a Kd,app of 150 pM and reduce the apparent DNA bending to less than 80 degrees. DNA footprinting analysis indicates that the nonspecific DNA binding activity of pRB is not involved in this effect. Our biochemical data suggest that transcriptional activation by E2F may involve DNA bending and that the reversal of bending upon binding of pRB may turn E2F into a repressor.
Huber
H E
HE
Department of Cancer Research, Merck Research Laboratories, West Point, Pennsylvania 19486.
Goodhart
P J
PJ
Huang
P S
PS
D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002846:Chromatography, Affinity; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D050684:E2F Transcription Factors; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D009838:Oligodeoxyribonucleotides; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D004247:DNA; D003850:Deoxyribonuclease I