Common and variable contributions of Fis residues to high-affinity binding at different DNA sequences

被引:20
|
作者
Feldman-Cohen, LS
Shao, YP
Meinhold, D
Miller, C
Colón, W
Osuna, R
机构
[1] Univ Albany, Dept Biol Sci, Albany, NY 12222 USA
[2] CUNY Coll Staten Isl, Dept Chem, Coll Staten Isl, Staten Isl, NY 10314 USA
[3] CUNY Coll Staten Isl, Dept Chem, Macromol Assemblies Inst, Staten Isl, NY 10314 USA
[4] Rensselaer Polytech Inst, Dept Chem & Chem Biol, Troy, NY 12180 USA
关键词
D O I
10.1128/JB.188.6.2081-2095.2006
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Fis is a nucleoid-associated protein that interacts with poorly related DNA sequences with a high degree of specificity. A difference of more than 3 orders of magnitude in apparent K-d values was observed between specific (K-d, similar to 1 to 4 nM) and nonspecific (K-d, similar to 4 mu M) DNA binding. To examine the contributions of Fis residues to the high-affinity binding at different DNA sequences, 13 alanine substitutions were generated in or near the Fis helix-turn-helix DNA binding motif, and the resulting proteins were purified. In vitro binding assays at three different Fis sites (fis P 11, hin distal, and lambda attR) revealed that R85, T87, R89, K90, and K91 played major roles in high-affinity DNA binding and that R85, T87, and K90 were consistently vital for binding to all three sites. Other residues made variable contributions to binding, depending on the binding site. N84 was required only for binding to the X attR Fis site, and the role of R89 was dramatically altered by the lambda attR DNA flanking sequence. The effects of Fis mutations on fis P 11 or hin distal site binding in vitro generally correlated with their abilities to mediate fis P repression or DNA inversion in vivo, demonstrating that the in vitro DNAbinding effects are relevant in vivo. The results suggest that while Fis is able to recognize a minimal common set of DNA sequence determinants at different binding sites, it is also equipped with a number of residues that contribute to the binding strength, some of which play variable roles.
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页码:2081 / 2095
页数:15
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