D. Chaudhary et Dm. Miller, THE C-MYC PROMOTER-BINDING PROTEIN (MBP-1) AND TBP BIND SIMULTANEOUSLY IN THE MINOR-GROOVE OF THE C-MYC P2 PROMOTER, Biochemistry, 34(10), 1995, pp. 3438-3445
The c-myc promoter binding protein (MBP-1) is a DNA binding protein wh
ich negatively regulates the expression of the human c-myc gene. MBP-1
binds to a sequence which overlaps; the binding site for the general
transcription factor TBP, within the c-myc P2 promoter region. Since T
BP binds in the minor groove, MBP-1 might inhibit c-myc transcription
by preventing the formation of a functional preinitiation complex. In
support of this hypothesis, we have demonstrated that MPB-1 is a minor
groove binding protein. In order to characterize MBP-1 binding, we su
bstituted A-T base pairs in the MBP-1 binding site with I-C base pairs
, which changes the major groove surface without altering the minor gr
oove surface. This substitution did not inhibit the sequence-specific
binding of MBP-1 and TBP. On the other hand, G-C to I-C substitution w
ithin the MBP-1 binding site alters the minor groove and prevents MBP-
1 binding. Competitive electrophoretic mobility shift assays were used
to show that berenil, distamycin, and mithramycin, all of which bind
in the minor groove, compete with MBP-1 for binding to the MPB-1 bindi
ng site. These minor groove binding ligands also effectively inhibit t
he simultaneous DNA binding activity of both MBP-1 and TBP. We conclud
e that both MBP-1 and TBP can bind simultaneously in the minor groove
of the TATA motif on the c-myc P2 promoter. This suggests that MBP-1 m
ay negatively regulate c-myc gene expression by preventing efficient t
ranscription initiation.