Andera L, Geiduschek E P
Department of Biology, University of California, San Diego, La Jolla 92093-0634.
J Bacteriol. 1994 Mar;176(5):1364-73. doi: 10.1128/jb.176.5.1364-1373.1994.
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.
分析了噬菌体SPO1编码的II型DNA结合蛋白TF1的羧基末端氨基酸在DNA结合中的作用。构建了在第96、97和98位氨基酸处截断正常99个氨基酸的TF1的链终止突变，以及在第97位氨基酸处用半胱氨酸、精氨酸和丝氨酸取代苯丙氨酸以及在第99位氨基酸处用色氨酸取代赖氨酸的错义突变。通过凝胶阻滞以及DNase I和羟基自由基足迹分析了所得蛋白质与5-(羟甲基)尿嘧啶DNA中一个合成的44碱基对结合位点、更大的SPO1 [含5-(羟甲基)尿嘧啶] DNA片段中的结合位点以及含胸腺嘧啶的同源DNA的结合情况。我们得出结论，直至并包括第97位氨基酸处的苯丙氨酸在内的C末端尾巴对于DNA结合至关重要，并且两个C末端氨基酸，即第98和99位氨基酸，参与了与DNA结合的TF1二聚体之间的蛋白质-蛋白质相互作用。
