Fig. 5

Binding specificity of TBP to the Pr77 sequence by EMSA competition. EMSA was carried out after preincubation of 0.125 nM ³²P-dsPr77 with 1.58 μM rTBP and with different concentrations (as indicated, fold excess ranging from 5 to 500 times) of unlabelled DNA competitors: a dsPr77 sequence and APT (aptamer mixture) and b poly(dI-dC) (commercial dI-dC polymer). As a negative control, reactions were carried out without protein (FP lane, Free Probe) or without competitor (B lane, Binding reaction). Reactions were performed at 37 °C for 30 min and resolved in 6% polyacrylamide native gels. In a and b, the asterisks indicate the free form that ³²P-dsPr77 adopts, and the black arrowhead indicates the formed complexes of higher molecular weight, which decrease in amount as the competitor concentration increases. c Fitting the competition kinetics data to a four-parameter logistic model with each competitor. Quantification was performed using ImageQuant software (Pharmacia)