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Journal of Bacteriology, March 2006, p. 2081-2095, Vol. 188, No. 6
0021-9193/06/$08.00+0     doi:10.1128/JB.188.6.2081-2095.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Common and Variable Contributions of Fis Residues to High-Affinity Binding at Different DNA Sequences

Leah S. Feldman-Cohen,2 Yongping Shao,1 Derrick Meinhold,3 Charmi Miller,3 Wilfredo Colón,3 and Robert Osuna1*

Department of Biological Sciences, University at Albany, 1400 Washington Avenue, Albany, New York 12222,1 Department of Chemistry, College of Staten Island and Macromolecular Assemblies Institute of the City, University of New York, Staten Island, New York 10314,2 Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, New York 121803

Received 27 October 2005/ Accepted 21 December 2005

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 Kd values was observed between specific (Kd, ~1 to 4 nM) and nonspecific (Kd, ~4 µM) 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 II, 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 {lambda} 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 II 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 DNA-binding 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.

* Corresponding author. Mailing address: Department of Biological Sciences, University at Albany, 1400 Washington Avenue, Albany, NY 12222. Phone: (518) 591-8827. Fax: (518) 442-4767. E-mail: osuna{at}albany.edu.

Journal of Bacteriology, March 2006, p. 2081-2095, Vol. 188, No. 6
0021-9193/06/$08.00+0     doi:10.1128/JB.188.6.2081-2095.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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