Three different polypeptides are necessary for DNA binding of the mammalian heteromeric CCAAT binding factor.
Full-length cDNA clones for the CBF-A and CBF-B subunits of the CCAAT binding mammalian heteromeric transcription factor (CBF) have previously been isolated from both rat and mouse. Whereas recombinant CBF-B binds to DNA after complementation with a highly purified CBF-A fraction, recombinant CBF-A was unable to bind to DNA after complementation with either purified CBF-B or recombinant CBF-B. However, when recombinant CBF-A, synthesized as a fusion protein with glutathione S-transferase was denatured together with a highly purified fraction containing CBF-A in the presence of 5.5 M guanidine hydrochloride and subsequently renatured, the recombinant CBF-A bound to DNA after complementation with CBF-B. This binding of recombinant CBF-A could not be detected if recombinant CBF-A was not mixed during the denaturation-renaturation process together with the purified fraction containing the 32-kDa CBF-A. Using a Southwestern blot we demonstrated that a polypeptide of approximately 40 kDa, present in the purified CBF-A fraction, bound to DNA after complementation with both recombinant CBF-A and CBF-B. After fractionation of the purified CBF-A preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a species of approximately 40 kDa was eluted from the gel and shown to have DNA binding activity after complementation with both recombinant CBF-A and CBF-B. Our results indicate that a third polypeptide, designated CBF-C, forms a tight complex with CBF-A. Together with CBF-A and CBF-B, CBF-C is required for the DNA binding activity of CBF.
Maity
S N
SN
Department of Molecular Genetics, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
Sinha
S
S
Ruteshouser
E C
EC
de Crombrugghe
B
B
D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008099:Liver / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D011489:Protein Denaturation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA