In a previous study we used 60 experimentally defined Fis binding sites to construct a highly detailed and reliable model of Fis binding to DNA [5]. When we searched DNA sequences for Fis sites using this information-theory based weight matrix model, we observed Fis sites spaced 11 base pairs apart in the DNA inversion systems hin, gin, and min (see Fig. 5 in reference [5]). A search of the entire E. coli genome shows that Fis sites are frequently separated by 11 bases (Fig. 1). Although the same number of sites are found in a genome-sized equiprobable random sequence, this does not mean that the sites are not functional. Many binding sites have just the amount of information ( Rsequence) needed for them to be found in the genome ( Rfrequency) [18]. According to Shannon's principles [47], a well-coded communications system looks like random noise from the outside, and Fis site pairs may follow this principle. So the spike of sites coming in pairs is caused by the pattern of Fis itself; it is intrinsic to the Fis model. We cannot tell if the pattern evolved because Fis sites were required to overlap or whether Fis sites overlap because the pattern evolved. The peaks of Fis sites every three bases in the genomic scan come from coding effects. The peak of spacing at 8 bases and excess sites at spacings of 1 and 3 bases were only noticed during revisions of this paper; we do not understand their significance.
Since B-form DNA twists every 10.6 base pairs, the sites should be on the same side of the DNA. While it is possible for two adjacent proteins to bind simultaneously by a subtle interleaving of their DNA contacts, as in the case of RNA polymerases [48,49], it seems more likely that in this case they will compete for binding in the major groove since after an 11 base shift the sequence logo shows that the predominant G at -7 corresponds to the G at +4 and the C at -4 corresponds to the C at +7(red arrows in Fig. 2). Competition between these internally redundant patterns [9] would allow Fis to change the point at which the DNA is bent. Perhaps this is important for inversion.
In contrast to the 11 base spacing described above,
in bacteriophage P1 cin,
bacteriophage cin,
E. coli e14 pin sites,
and in the genome scan
(Fig. 1),
pairs of Fis sites are observed at spacings of
7 bases.
This would place the Fis dimers
122
apart on B-DNA
(
.
After a 7 base shift, the sequence logo shows
that the predominant G at -7 would match the A/T/g/c
of the minor groove on the opposite
face of the DNA at coordinate 0, while the C/T at -4 would match the T/C at
+3 and the A/G at -3 would match the G/A at +4(green arrows in Fig. 2).
This allows for the possibility that the
two proteins bind at the same time,
which might also be important to the
function of these regions.
To investigate the consequences of two Fis molecules binding to nearby sites, we constructed 3 dimensional models (Fig. 3). We found that two Fis proteins bound to sites separated by 11 base pairs might strongly interpenetrate. In contrast, a 7 base pair separation might only have a minimal van der Waals force conflict between the two central D helices. This might be accommodated for by flexibility of the DNA-protein complex, given that there is some uncertainty as to how Fis binds DNA. The models suggest that 11-base separated Fis molecules would compete for binding but that a 7-base separation might allow simultaneous binding.
These ideas are supported by the preliminary observation that synthetic DNA containing either the hin proximal or medial Fis sites are bound by Fis in electrophoretic mobility gel shift assays [5]. When these overlapping sites were together on the same fragment with a spacing of 11 bases only one band shift was observed, suggesting that only one of the sites can be bound at a time. To test whether this is the case requires using high concentrations of Fis and strong Fis sites to ensure that both sites would be bound if that were possible.