DETERMINANTS OF AFFINITY AND MODE OF DNA-BINDING AT THE CARBOXY-TERMINUS OF THE BACTERIOPHAGE-SPO1-ENCODED TYPE-II DNA-BINDING PROTEIN, TF1

The role of the carboxy-terminal amino acids of the bacteriophage SPO1 -encoded type II DNA-binding protein, TF1, in DNA binding was analyzed . Chain-terminating mutations truncating the normally 99-amino-acid TF 1 at amino acids 96, 97, and 98 were constructed, as were missense mut ations substituting cysteine, arginine, and serine for phenylalanine a t amino acid 97 and tryptophan for lysine at amino acid 99. The bindin g of the resulting proteins to a synthetic 44-bp binding site in 5-(hy droxymethyl) uracil DNA, to binding sites in larger SPO1 [5-(hydroxyme thyl)uracil-containing] DNA fragments, and to thymine-containing homol ogous DNA was analyzed by gel retardation and also by DNase I and hydr oxy radical footprinting. We conclude that the C tail up to and includ ing phenylalanine at amino acid 97 is essential for DNA binding and th at the two C-terminal amino acids, 98 and 99, are involved in protein- protein interactions between TF1 dimers bound to DNA.