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 TF1 at amino acids 96, 97, and 98 were constructed, as were missense mutations substituting cysteine, arginine, and serine for phenylalanine at amino acid 97 and tryptophan for lysine at amino acid 99. The binding of the resulting proteins to a synthetic 44-bp binding site in 5-(hydroxymethyl)uracil DNA, to binding sites in larger SPO1 [5-(hydroxymethyl)uracil-containing] DNA fragments, and to thymine-containing homologous DNA was analyzed by gel retardation and also by DNase I and hydroxy radical footprinting. We conclude that the C tail up to and including phenylalanine at amino acid 97 is essential for DNA binding and that the two C-terminal amino acids, 98 and 99, are involved in protein-protein interactions between TF1 dimers bound to DNA.
Andera
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Department of Biology, University of California, San Diego, La Jolla 92093-0634.
Geiduschek
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EP
D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004279:DNA, Viral / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C059361:TF1 DNA-binding protein, Bacteriophage SPO1; D014764:Viral Proteins