Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 380, Issue 2, Pages 327-339Publisher
ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2008.04.075
Keywords
Fis; nucleoid-associated protein; DNA binding; ethylation interference; missing base contact
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Funding
- NIGMS NIH HHS [GM52051, R01 GM052051-09, R01 GM052051, R29 GM052051] Funding Source: Medline
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Fis (factor for inversion stimulation) is a nucleoid-associated protein in Escherichia coli and other bacteria that stimulates certain site-specific DNA recombination events, alters DNA topology, and serves as a global gene regulator. DNA binding is central to the functions of Fis and involves a helix-turn-helix DNA binding motif located in the carboxy-terminal region. Specific DNA binding is observed at a number of sites exhibiting poorly related sequences. Such interactions require four critical base pairs positioned -7, -3, +3, and +7 nucleotides relative to the central nucleotide of a 15-bp core-binding site. To further understand how Fis interacts with DNA, we identified the positions of 14 DNA phosphates (based on ethylation interference assays) that are required for Fis binding. These are the 5' phosphates of the nucleotides at positions -8, -7, -6, +1, +2, +3, and +4 relative to the central nucleotide on both DNA strands. Another five phosphates located in the flanking regions from positions +10 through +14 can serve as additional contact sites. Using a combination of biochemical approaches and various mutant Fis proteins, we probed possible interactions between several key Fis residues and DNA bases or phosphates within a high-affinity binding site. We provide evidence in support of interactions between the R85 Fis residue and a highly conserved guanine at position - 7 and between T87 and the critical base pairs at -3 and +3. In addition, we present evidence in support of interactions between N84 and the phosphate 5' to the base at +4, between R89 and the - 7 phosphate, between T87 and the +3 and +4 phosphates, and between K90 and the +3 phosphate. This work provides functional evidence for some of the most critical interactions between Fis and DNA required for a high binding affinity and demonstrates the large contribution made by numerous phosphates to the stability of the Fis-DNA complex. (C) 2008 Elsevier Ltd. All rights reserved.
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