The Escherichia coli protein Fis is remarkable for its ability to interact specifically with DNA sites of highly variable sequences. The mechanism of this sequence-flexible DNA recognition is not well understood. In a previous study, we examined the contributions of Fis residues to high-affinity binding at different DNA sequences using alanine-scanning mutagenesis and identified several key residues for Fis-DNA recognition. In this work, we investigated the contributions of the 15-bp core Fis binding sequence and its flanking regions to Fis-DNA interactions. Systematic base-pair replacements made in both half sites of a palindromic Fis binding sequence were examined for their effects on the relative Fis binding affinity. Missing contact assays were also used to examine the effects of base removal within the core binding site and its flanking regions on the Fis-DNA binding affinity. The results revealed that: (1) the -7G and +3Y bases in both DNA strands (relative to the central position of the core binding site) are major determinants for high-affinity binding; (2) the C(5) methyl group of thymine, when present at the +4 position, strongly hinders Fis binding; and (3) AT-rich sequences in the central and flanking DNA regions facilitate Fis-DNA interactions by altering the DNA structure and by increasing the local DNA flexibility. We infer that the degeneracy of specific Fis binding sites results from the numerous base-pair combinations that are possible at noncritical DNA positions (from -6 to -4, from -2 to +2, and from +4 to +6), with only moderate penalties on the binding affinity, the roughly similar contributions of -3A or G and +3T or C to the binding affinity, and the minimal requirement of three of the four critical base pairs to achieve considerably high binding affinities.

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 , ϳ1 to 4 nM) and nonspecific (K d , ϳ4 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 II, hin distal, and 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 attR Fis site, and the role of R89 was dramatically altered by the 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 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.Fis (factor for inversion stimulation) is the most abundant nucleoid-associated protein during the logarithmic growth phase in rapidly growing Escherichia coli cells (3, 6). However, during mid-to late-logarithmic growth, the intracellular levels of Fis decrease over 500-fold and become nearly imperceptible during the stationary phase. The impact of Fis on cell physiology is widespread. As a nucleoid-associated protein, it is able to interact with a large number of DNA sites to alter DNA topology (66,67). Numerous genes are subject to positive or negative regulation by Fis directly or indirectly (6,10,19,22,30,49,56,61,(73)(74)(75). In the case of the promoters rrnB P1 and proP P2, Fis binding to sites centered at positions Ϫ71 and Ϫ41, respectively, directly stimulates transcription by contacting the C-terminal domain of the RNA polymerase ␣ subunit (␣-CTD) (9, 43). In the case of the fis promoter (fis P), Fis binding to sites I and II, centered at positions ϩ25 and Ϫ44, respectively, negatively autoregulates the fis operon by hindering RNA polymerase binding (6, 50, 58). As its name suggests, Fis also stimulates site-specific DNA inversion involving the Hin, Gin, and Cin family of recombinases (24,32,35). During Hin-mediated DNA inversion, Fis binds to two high-affinity DNA sites (hin-proximal and -distal sites) in a 60-bp recombinational enhancer region and interacts with two DNA-bound Hin recombinases to form a nucleoprotein complex intermediate required for efficient DNA strand cleavage and inversion (25,33). Bacteriophage DNA excis...

SUMMARYFis is a nucleoid-associated protein in E. 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 fourteen 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.