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

Journal of Bacteriology
Leah S Feldman-CohenRobert Osuna

Abstract

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, approximately 1 to 4 nM) and nonspecific (Kd, approximately 4 microM) 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 i...Continue Reading

References

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Citations

Apr 16, 2010·Molecular Biology and Evolution·Estelle CrozatDominique Schneider
Apr 17, 2010·Genes & Development·Stefano StellaReid C Johnson
Jan 8, 2008·Journal of Molecular Biology·Yongping ShaoRobert Osuna
May 15, 2007·Journal of Molecular Biology·Stacey N PetersonNorbert O Reich
Feb 1, 2011·Protein Science : a Publication of the Protein Society·Virginia A MuñizWilfredo Colón
Oct 17, 2019·Nucleic Acids Research·Stephen P HancockReid C Johnson
May 2, 2020·Acta Crystallographica. Section F, Structural Biology Communications·Juan ZhouYuhui Dong
Mar 25, 2020·The Biochemical Journal·Soumyananda ChakrabortiGary J Sharples

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