Fig. 4. DNA-binding properties of TDF. (A) Consensus binding sequence of TDF. After five cycles of immunoselection of TDF-bound PCR amplified random oligonucleotides, the recovered DNA was cloned into a T-vector and sequenced. Percentages indicate frequencies at which each nucleotide appeared at a given position from 1 to 14 in the total of 54 sequenced clones. The second line represents the deduced consensus sequence. (B) Electrophoretic mobility shift assay for DNA binding of TDF. Ten fmoles of ³²P-labeled DNA probe and 25 ng of His-TDF in 10 µl of binding mixture were incubated at 25°C for 60 minutes. The DNA-TDF complex (b) was separated from the free probe (f) by electrophoresis. Lanes 1 and 2, binding reaction without any competitor; lane 1 received a mock-purified fraction from bacteria carrying an empty vector in place of His-TDF. In lanes 3-8, samples contained fold excess amounts of the unlabeled competitor DNA: either the functional TDS (lanes 3-5), or the mutant TDMS (lanes 6-8) sequence.