FIG. 9.

Overexpression of C/EBPβ disrupts formation of the TBP-TATA complex on the HPV-16 URR in HeLa cells. ³²P-labeled oligonucleotide 16TATA was used in EMSAs with HeLa cell nuclear extracts. Binding reactions were carried out with extracts from nontransfected cells in the absence (/) (lanes 2, 7, 13, and 16) and presence (lanes 3 to 6) of unlabeled competitor oligonucleotides. Unlabeled competitor oligonucleotides 16TATA and 16TATA-M1 were used in a 10- or 50-fold molar excess, as indicated above the lanes. Anti-human TBP antibodies were included in the binding reaction with nuclear extract from nontransfected HeLa cells (lane 8). Binding reactions were also carried out with nuclear extracts from HeLa cells transfected with increasing amounts of CMV-C/EBPβ (lanes 9 and 10), CMV-LIP (lanes 11 and 12), or pcDNAI (lanes 14 and 15) as indicated above the lanes. In lane 1, a standard binding reaction was carried out with HeLa cell nuclear extract and ³²P-labeled probe 18TATA. On the left side (for probe 18TATA), a specific DNA-protein complex is indicated by the I (marked by an arrow); numerals II to IV indicate nonspecific DNA-protein complexes. On the right side (for probe 16TATA), a specific DNA-protein complex is indicated by 16I (marked by an arrow); 16II to 16VII indicate nonspecific DNA-protein complexes. F, free probe 18TATA (lane 1) or free probe 16TATA (lanes 2 to 16). trans., transfected.