Originally discovered in the bacteriophage Mu DNA inversion system gin, Fis (Factor for Inversion Stimulation) regulates many genetic systems. To determine the base frequency conservation required for Fis to locate its binding sites, we collected a set of 60 experimentally defined wild-type Fis DNA binding sequences. The sequence logo for Fis binding sites showed the significance and likely kinds of base contacts, and these are consistent with available experimental data. Scanning with an information theory based weight matrix within fis, nrd, tgt/sec and gin revealed Fis sites not previously identified, but for which there are published footprinting and biochemical data. DNA mobility shift experiments showed that a site predicted to be 11 bases from the proximal Salmonella typhimurium hin site and a site predicted to be 7 bases from the proximal P1 cin site are bound by Fis in vitro. Two predicted sites separated by 11 bp found within the nrd promoter region, and one in the tgt/sec promoter, were also confirmed by gel shift analysis. A sequence in aldB previously reported to be a Fis site, for which information theory predicts no site, did not shift. These results demonstrate that information analysis is useful for predicting Fis DNA binding.

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The RepA protein from bacteriophage P1 binds DNA to initiate replication. RepA covers one face of the DNA and the binding site has a completely conserved T that directly faces RepA from the minor groove at position +7. Although all four bases can be distinguished through contacts in the major groove of B-form DNA, contacts in the minor groove cannot easily distinguish between A and T bases. Therefore the 100% conservation at this position cannot be accounted for by direct contacts approaching into the minor groove of B-form DNA. RepA binding sites with modified base pairs at position +7 were used to investigate contacts with RepA. The data show that RepA contacts the N3 proton of T at position +7 and that the T=A hydrogen bonds are already broken in the DNA before RepA binds. To accommodate the N3 proton contact the T(+7 )/A(+7)((')) base pair must be distorted. One possibility is that T(+7) is flipped out of the helix. The energetics of the contact allows RepA to distinguish between all four bases, accounting for the observed high sequence conservation. After protein binding, base pair distortion or base flipping could initiate DNA melting as the second step in DNA replication.

The DNA-binding protein Fis frequently uses pairs of sites 7 or 11 base pairs (bp) apart. Two overlapping Fis sites separated by 11 bp are found in the Escherichia coli origin of chromosomal replication. Only one of these sites is bound by Fis at a time, so the structure is a molecular flip-flop that could direct alternative firing of replication complexes in opposite directions. Alternatively, the flip-flop could represent part of an on-off switch for replication. Because they can be used to create precise switched states, molecular flip-flops could be used as the basis of a novel molecular computer.