FIG. 9.

T antigen increases formation and prevents the dissociation of the TA complexes formed on the hsp70 TATA element. (A) Determination of the association rate of the TA complex in the presence and absence of T antigen. An EMSA was performed to determine the rate of association of the TA complex on the ³²P-labeled Sp1-hsp70 TATA promoter in the presence or absence of T antigen. Binding reaction mixtures were prepared, and at different time points after mixing, samples were loaded on a running gel. The intensity of the bands representing the TA complex was quantitated with a Molecular Dynamics PhosphorImager and plotted as amount of TA complex formed over time. (B) Determination of the dissociation rate of the TA complex in the presence and absence of T antigen. Binding reaction mixtures similar to those described for panel A were incubated for 1 h to bring the binding to equilibrium. Then an excess of unlabeled Sp1-hsp70 TATA promoter DNA was added as a competitor. At various time points after addition of the competitor, samples were removed and loaded on a running gel. The intesity of the bands representing the TA complex was quantitated with a PhosphorImager and plotted as a percentage of the intensity of the band at equilibrium.