Genetic implications of Fis flip-flops

The Escherichia coli tyrT promoter has three Fis sites separated by 20 and 31 base pairs. This separation corresponds to our 23 base pair separated control experiment (Fig. 4 and Fig. 5), in which two Fis molecules bind independently. The separation in tyrT is sufficient for three Fis dimers to simultaneously position themselves on the same face of the DNA to cooperatively bind an $0.006 \pm 0.001$CTD subunit and activate transcription of stable RNA promoters [51]. In addition to this activation mechanism, which is based on separated sites, Fis may also have evolved another control mechanism that uses overlapping sites.

When we scanned our Fis individual information model across various sequences, we discovered 7 and 11 spacings at DNA inversion regions, the fis, nrd, and ndh promoters, and at dif, E. coli oriC and $\gamma$ att [10,5]. In the latter three systems, sequence walkers for Fis sites overlap binding sites of other proteins in biologically significant places, so we do not think that Fis sites appear at this spacing merely because of the internal redundancy of the site. Scanning with the Fis weight matrix also reveals two strong Fis sites previously identified in oriC at coordinates 202 and 213 although only one was thought to be bound [52,53,54]. Footprinting data from three different groups show protection covering one, the other and both sites (Fig. 6).

We confirmed both oriC Fis sites individually by a gel mobility shift experiment (Fig. 7). Because of our previous work on Fis promoters [5], we knew that we could use sequence walkers [10] to engineer the sites. This way we could know that we had destroyed one site without affecting the other and without creating new sites [48]. When both sites were mutated (oligo nn), no shift was observed until a high Fis concentration (1000 nM) was used. We take this concentration to represent non-specific binding for all four oligos used in the experiment. When the left (oligo no) or right (oligo on) Fis sites were mutated, only a single shifted band was observed. This confirms our prediction that both sites can bind Fis. In the on control a high band appeared at the highest Fis concentration. From the lower concentration lanes, we know that there is one specific binding site and, apparently, at this extremely high concentration a second Fis molecule binds nonspecifically on this particular DNA sequence. This additional band may be explained by the existence of a number of weak (< 0 bit) sites that can bind DNA at high Fis concentration. In any case this band is absent in the experimental lane (oo).

The experiment also shows that the left Fis site, which is closer to DnaA R2 (oligo on, 8.4 bits) binds slightly less strongly than the one closer to DnaA R3 (oligo no, 9.1 bits) confirming the respective individual information contents, which differ by about 1 bit. When both sites were wild-type (oligo oo) Fis binding was also observed but no supershift was visible. We conclude that only one Fis molecule can bind at a time between DnaA R2 and DnaA R3 of oriC.

The oo oligo can be bound in two distinct ways, so its association constant to Fis should be the sum of the two individual site association constants. This effect may have practical applications since creating overlapping binding sites will double the sensitivity of a biological detection system [55].

The two Fis sites at oriC fit exactly between the R2 and R3 DnaA sites and have similar individual information contents, suggesting that their binding energies are similar [56], so in the absence of other effects Fis could occupy them for nearly equal fractions of the time as a molecular flip-flop. The two states have not been recognized before because DNA footprinting only shows one predominant state or shows both states simultaneously, and such footprints have hitherto been interpreted as representing single sites. That is, the macroscopic experiments did not reveal that there are two distinct binding modes at dual Fis sites.

Our results resolve two previously conflicting reports. Gille et al found that Fis binding and DnaA binding at R3 are mutually exclusive [52] but Margulies and Kaguni found that they could bind concurrently [57]. The controversy may be resolved by noting that different experimental techniques were used and that, because consensus sequences were being used, it was not clear that there are two Fis sites [22]. The experiments by Gille et al were DNase I footprints, which show protection of the entire R2-R3 region when Fis is bound prior to DnaA, as would be expected from a mixture of two states. The experiments by Margulies and Kaguni were footprints and gel shifts. If DnaA binds to R3, Fis might be blocked at position 213 but Fis could still bind at the other site at position 202. It is possible that both experiments produced valid data but for different states of the flip-flop.

If binding by DnaA and Fis are mutually exclusive [52], then the position of a Fis-induced DNA bend could be controlled by DnaA and the binding of DnaA could be controlled by Fis. During nutritional upshifts when there is a high Fis concentration [4], occupancy of one Fis site should ensure that only one DnaA site is available at a time. DnaA directs the loading of the DnaB helicase which, in turn, determines the orientation of the DNA polymerase or DnaG primase [58] and therefore the direction of replication [59,60,61,12,62]. Since the Fis flip-flop probably controls which of the two oppositely oriented DnaA sites can be bound, it may control the alternative firing of replication complexes in opposite directions [63]. This appears to be consistent with the divergent directions of DNA initiation observed in this region, as shown in Fig. 6 [64]. Indeed, the absence of Fis leads to asynchronous replication [65], and at high temperatures fis null mutants have been shown to form filamentous cells and have aberrant nucleoid segregation [66]. Although Fis is pleiotropic, these and other observations [67,68] are consistent with Fis being required for proper replication initiation. However, initiation using purified components in the absence of Fis is bidirectional [59,69]. One possibility is that in the absence of Fis, loading orientation is random [69] and that initiation in vivo fails unless the complexes are oppositely oriented. This should occur $R_{frequency} = \log_2{812} = 9.7$25% of the time, and indeed when initiating molecules were counted, only 36% formed SSB bubbles [59]. Since there may be other explanations for these data, further experiments will be needed to determine how the Fis flip-flop is involved in initiation.

A general model for how DnaA is involved in origin replication has been proposed [70,71] in which the R1 DnaA site is thought to be involved in opening the adjacent AT rich 13 mer region. That model does not include the two competing Fis sites demonstrated in this paper. To combine the models, one possibility is that the genetic structure at R2-R3 is only functional at nutritional upshifts when there is a high concentration of Fis in the cell [4]. Under these circumstances coordination of replication fork firing may be critical to start the first or subsequent rounds of replication correctly.

Are these proposals supported by mutations in the Fis site(s)? An experiment by Weigel et al was intended to destroy `the' Fis site between R2 and R3 [72], however analysis by sequence walkers shows that the oriC131 AACTCAA to ATGTGTA mutation decreased the left Fis site (at coordinate 202) from 8.4 to 3.1 bits, while leaving the right site at 213 unchanged (analysis not shown). When placed in the E. coli chromosome, the mutant shows a moderate change of asynchrony of initiation by flow cytometry. Unfortunately, the mutation also created an 8.9 bit DnaA site which makes the experimental results difficult to interpret. (This 8.9 bit site also was created in earlier experiments [53].) In another experiment, replacing six bases between R2 and R3 by a BamHI site decreased oriC dependent plasmid transformation by 57 fold [73]. Sequence walker analysis (not shown) indicates that this mutation destroys both Fis sites (< 2 bits) while leaving the R2 and R3 DnaA sites intact. A 10 base pair insertion at coordinate 203 (presumably between bases 203 and 204) destroyed the MPE (Methidiumpropyl-EDTA-Fe⁺⁺) footprint of the Fis site at 202 but the site at 213 still showed an MPE footprint [54]. This same insert reduced transformation frequency of an oriC plasmid into a polA strain. These results suggest that the Fis flip-flop is important for replication from oriC.

When does the flip-flop state change? In vivo footprinting shows that during the cell cycle DnaA sites R1, R2 and R4 are bound, but R3 is not occupied [74]. R3 becomes occupied at initiation of DNA replication [75]. R3 is also bound by DnaA more weakly than R2 in vitro [76,77]. This is consistent with the information measures which suggest that R3 is 13.5 - 12.1 = 1.4 bits or at least 2^1.4 = 2.6 fold weaker (Fig. 6). In the absence of other processes, R3 should be bound less frequently. We suggest that for the majority of the cell cycle DnaA bound to R2 blocks Fis at position 202, which allows Fis to bind at 213 and which, in turn, entirely blocks R3 from being bound by DnaA. Replication initiation may temporarily alter the flip-flop state, exposing R3. These data suggest the alternative hypothesis that the flip-flop is part of an on-off switch controlling initiation in the presence of Fis, especially during nutritional upshift [4].

Closely spaced sites are often bound cooperatively, as in the classical example of T4 gene 32 autogenous regulation [78], and even at overlapping LexA sites on opposite faces of the DNA [79,80]. In contrast, Fis represents the unusual situation where a protein competes with itself by binding at overlapping positions. Self-occlusion has been observed in artificial constructs, where one ribosome is apparently blocked by the presence of another ribosome bound nearby [81], by polymerases at promoters [48,82,49,83], and in enzyme complexes [84]. An interplay of factors may be typical of complex flip-flop mechanisms. For example, as many as five Fis sites are likely to be in $\gamma$ att. Two of these are spaced 11 base pairs apart, with one of them overlapping an Xis site [10]. Likewise, at the E. coli dif locus, where the XerC and XerD site-specific recombination proteins bind [85], one finds an overlapping set of 7 weak Fis sites; three of these are separated by 11 bases (data not shown).

The positioning of Fis binding sites relative to one another and to the binding sites of other proteins therefore appears to be key for the ability of Fis to perform many diverse functions. Fis has evolved a transcriptional activation mode in which sites are on the same face of the DNA and are sufficiently far apart to be bound simultaneously [51]. Fis may also have specifically evolved to allow for two competitive binding modes. When the sites are on the same face of DNA (11 bp apart), a single Fis molecule could disengage and rebind to move the bend location between two possible places without changing the overall direction of the DNA. When sites are on nearly opposite faces (7 bp apart), shifting a Fis dimer molecule would cause the bend direction to change by 122 $4.0 \pm 0.4$. How these cogs fit into the larger picture of pleiotropic Fis functions remains to be determined. However, an 11 base pair shift of a bend would have dramatic effects on the oriC DNA initiation complex structure such as the ones suggested by Messer et al [54,86].