Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 364 (4), 777-98

Mechanism of Chromosome Compaction and Looping by the Escherichia Coli Nucleoid Protein Fis


Mechanism of Chromosome Compaction and Looping by the Escherichia Coli Nucleoid Protein Fis

Dunja Skoko et al. J Mol Biol.


Fis, the most abundant DNA-binding protein in Escherichia coli during rapid growth, has been suspected to play an important role in defining nucleoid structure. Using bulk-phase and single-DNA molecule experiments, we analyze the structural consequences of non-specific binding by Fis to DNA. Fis binds DNA in a largely sequence-neutral fashion at nanomolar concentrations, resulting in mild compaction under applied force due to DNA bending. With increasing concentration, Fis first coats DNA to form an ordered array with one Fis dimer bound per 21 bp and then abruptly shifts to forming a higher-order Fis-DNA filament, referred to as a low-mobility complex (LMC). The LMC initially contains two Fis dimers per 21 bp of DNA, but additional Fis dimers assemble into the LMC as the concentration is increased further. These complexes, formed at or above 1 microM Fis, are able to collapse large DNA molecules via stabilization of DNA loops. The opening and closing of loops on single DNA molecules can be followed in real time as abrupt jumps in DNA extension. Formation of loop-stabilizing complexes is sensitive to high ionic strength, even under conditions where DNA bending-compaction is unaltered. Analyses of mutants indicate that Fis-mediated DNA looping does not involve tertiary or quaternary changes in the Fis dimer structure but that a number of surface-exposed residues located both within and outside the helix-turn-helix DNA-binding region are critical. These results suggest that Fis may play a role in vivo as a domain barrier element by organizing DNA loops within the E. coli chromosome.


Figure 1
Figure 1. Sequence-independent Fis binding to DNA
A. Model of a Fis-DNA complex. The two helix-turn-helix motifs, spaced by 25 Å, bind successive major grooves on one side of the DNA helix, forcing a bend into the double helix. The mobile N-terminal β-hairpin arms protrude from the side opposite to the DNA. The gold ribbon over the DNA backbone denotes the 21 bp minimal binding site. The structure of Fis in this and subsequent figures is from the K36E mutant, in which residues 10–98 that include the β-hairpin arms are resolved , and it is modeled onto DNA with an overall curvature of about 50°. B. Gel mobility shift assays of Fis binding to a 100 bp DNA fragment from phage λ Fis was added in 2-fold increments beginning at 0.15 nM (lane 2) and ending at 600 nM (lane 14). No Fis was added in lane 1. The locations of individual Fis-DNA complexes, the “coated” complex, the “low mobility complex” (LMC), and the unbound (Free) DNA are denoted. C. Same as panel B, except that a 149 bp fragment from the yeast actin gene was the DNA substrate. D. Fis binding isotherms of the complex bound by one or more Fis dimers (left curve, blue triangles), the coated complex containing 7 or more Fis dimers (middle curve, red squares), and the LMC complex (right curve, green circles) assembled on the actin DNA fragment. Points represent the mean and standard deviations obtained from ≥4 independent experiments. Hill coeficients for the first complex, the coated complex, and the LMC are 1.5, 1.5, and 3.5, respectively.
Figure 2
Figure 2. DNA cleavage by Fis-OP nuclease chimeras when bound nonspecifically
A. Model of Fis98-OP bound to DNA. The 1,10 phenanthroline copper moiety linked to the C-terminus (residue 98) of Fis through an acetamido linker is shown in red with the copper as an orange sphere. B. A 262 bp fragment from the 5′ end of the lacZ gene and 32P-labeled at the 5′ end of the coding strand was incubated with 0 and approximately 5, 10, and 20 nM Fis98-OP (lanes 2–5, 7–10). Lanes 1 and 6 are G chemical sequencing ladders . The left and right panels are the same samples electrophoresed for longer or shorter time periods, respectively, on the same gel to resolve most of the DNA length. C. DNase I and Fis98-OP cleavage. The fragment used in panel B was subjected to DNase I footprinting with 0, and approximately 1, 2.5, 5, 10, and 20 nM Fis (lanes 2–7). Lanes 8 and 9 are incubation with no protein or 10 nM Fis98-OP, respectively. Lane 1 is a G sequencing ladder. Arrowheads denote major Fis98-OP cleavage sites, and the vertical bar denotes a weak DNase I protected region by Fis about 150 bp into the lacZ coding region.
Figure 3
Figure 3. Single-DNA study of Fis-DNA interactions
A. Vertical magnetic tweezer setup. A single DNA molecule is tethered to the top surface inside the flow cell; a paramagnetic bead attached at the other end of the molecule provides a ‘handle’ to which controlled forces can be applied using permanent magnets on a translator. The bead is imaged using a 100x objective on a piezoelectric positioner (bottom panel), allowing a computer to track the bead in three dimensions. B. Force-extension curves for bare DNA (black) in buffer with glycerol and BSA (see text), and with Fis at 10 nm (light green) 20 nM (orange), 50 nM (dark green), 100 nM (violet) and 200 nM (blue) concentrations. At 10 nM, little effect is observed; at 20 nM, maximum compaction occurs. As concentration is increased further, a gradual reduction of compaction occurs. Over this (low) concentration range, all force-extension curves are entirely reversible, indicating no tendency for Fis to stabilize DNA crossings. C. Collapse of DNA against applied force driven by Fis. Naked DNA in buffer (open circles) and DNA incubated with 200 nM Fis (blue crosses) and 500 nM Fis (green circles) show reversible force-extension response over the full range of forces, but for 1 μM Fis concentration (orange diamonds) the Fis-DNA complex undergoes a collapse (horizontal dashed line) when force is reduced to 0.2 pN. For larger Fis concentrations of 6 μM (blue triangles), and 13 μM (red squares) similar collapse transitions occur at higher forces of 0.3 and 0.6 pN, respectively.
Figure 4
Figure 4. Step observation and analysis during collapse and subsequent reopening of Fis-DNA complexes
A. Extension versus time for a 10.5 kb DNA in 10 μM Fis solution and under a 0.5 pN constant force. The ~100 nm rapid fluctuation in the extension around its local average is due to thermal fluctuations. Steps down, and occasionally back up, are observed, with amplitudes in the 200 nm range. B. Reopening of the Fis-DNA complex following the collapse shown in A, driven by a force of 7 pN. Opening proceeds via a series of jump events of roughly 200 nm amplitude, consistent with reversal of the step-wise condensation shown in A. C. Collapse of a 10.5 kb DNA in 1 μM Fis solution, against a 0.2 pN force. Note the larger thermal noise amplitude relative to A, and also the relatively small initial extension. However, step-like features are still observable during condensation. D. Reopening of the Fis-DNA complex following the collapse shown in C, driven by a force of 4 pN; as in B, opening proceeds via a series of step events of roughly 200 nm size. E. Step size distribution obtained from closing traces from a series of 10 μM Fis experiments similar to that shown in A. The average step size is 225 nm. F. Step size distribution obtained from opening traces from a series of 10 μM Fis experiments similar to B. The average step size is 275 nm. G. Step size distribution obtained from closing traces from a series of 1 μM Fis experiments similar to C. The average step size is 250 nm; after correction for the low force involved, the DNA length involved in each step is roughly double the extension step observed. H. Step size distribution obtained from opening traces from a series of 1 μM Fis experiments similar to D. The average step size is 210 nm.
Figure 5
Figure 5. Stability of Fis-DNA complexes
A. Naked λ-DNA in buffer (open circles) was incubated with 50 nM Fis (filled squares). Then, the protein solution was replaced with protein-free buffer and incubated for 20 min, and the force-extension response was re-measured (open squares). The lack of any change indicates that Fis which is “coating” DNA does not spontaneously dissociate into buffer. B. Naked λ-DNA in buffer (open circles) was incubated with 6 μM Fis while held extended by a 10 pN force. Then, the protein solution was replaced by protein-free buffer; finally force was gradually reduced and extensions were measured (filled circles); Fis-induced collapse was observed at 0.3 pN, indicating that looping-competent Fis bound to DNA does not spontaneously dissociate into buffer. C. Naked λ-DNA in buffer (open circles) was incubated with 50 nM Fis, resulting in the expected high-force compaction effect (open triangles). Then, the protein solution was replaced by protein-free buffer containing 50 μg/ml nonspecific DNA fragments, which resulted in no change in the high-force compaction (filled triangles). Higher DNA fragment concentration of 250 μg/ml (filled circles) led to a shift of the force curve back towards that of naked DNA indicating that Fis may transfer to competitor DNA at sufficiently high concentration. D. In an experiment similar to that shown in C, initially naked DNA (open circles) was incubated with 5 μM Fis solution, resulting in the expected shift of the force-extension curve above 1 pN (filled squares; data for the force shift generated by 200 nM Fis is shown for comparison, filled triangles). After washing with 50 μg/ml DNA fragments the complex retained the force-extension behavior shifted relative to naked DNA (in accord with C), but did not undergo looping-condensation at low forces (open squares). Thus, DNA-bending Fis is stable in the presence of nonspecific DNA, but DNA-looping Fis can be removed (or quenched) by nonspecific DNA.
Figure 6
Figure 6. Effects of salt on Fis-DNA interactions
Naked λ-DNA (filled circles) undergoes a modest compaction at high forces, and collapse at low forces after incubation with 5 μM Fis in our standard-salt buffer (100 mM KGlu, triangles). In a second experiment using 5 μM Fis in high-salt buffer (500 mM KGlu, filled squares) the high-force compaction occurs but no collapse occurs at low force. Washing the sample with the standard-salt buffer (100 mM KGlu, filled circles) did not lead to looping-condensation. In a third experiment, DNA was first incubated with 5 μM Fis in 100 mM KGlu buffer, and then the protein solution was replaced with 500 mM KGlu protein-free buffer and finally force was reduced; the result was collapse at 0.5 pN (filled diamonds). Thus, salt concentration at the time of binding controls the ability for Fis-DNA complexes to undergo looping-condensation.
Figure 7
Figure 7. Behavior of single-site mutants of Fis
A. Surface representation of the Fis dimer in two orientations showing the locations of mutated residues studied. The two subunits of the dimer are in light and dark grey unless otherwise colored. Individual amino acid residues analyzed in this study are colored with respect to their looping and compaction activities: similar to wild-type (blue), looping defective (red), and DNA stiffening (green). Proteins containing substitutions at residues with asterisks also exhibit reduced DNA compaction. B. Deletion of N-terminal arm residues 2 to 26 does not affect looping-compaction. Naked λ-DNA (open circles) was incubated with 5 μM Fis mutant Δ(2-26) (filled circles). Although the DNA extensions in the high force range approach the naked DNA in this experiment, other experiments with this mutant exhibit a high force compaction shift similar to the wild-type. C. Fis residue Arg 71 is plays an important role in looping-collapse. Naked λ-DNA (open circles) was incubated with Fis mutant R71A (filled squares) with the result that high-force compaction was observed, but no low-force collapse occurred. In a separate experiment with R71K (open squares), high-force compaction and low-force looping-collapse were nearly indistinguishable from wild-type. In a third separate experiment, R71Y (open diamonds) generated high-force compaction and promoted looping-collapse at a higher threshold force than observed with wild-type Fis. D. Micromechanical properties of Fis mutants K36E, R76A, K90A, and G72D. Complexes on λ-DNA were formed with 5 μM of each mutant. The force-shift by Fis K36E (filled triangles) is nearly indistinguishable from the naked DNA (open circles), and no looping-condensation occurred. Mutants R76A (open triangles) and K90A (filled squares) exhibited no looping-condensation, but did generate a compaction force-shift. Fis G72D (filled diamonds) exhibits both bending-compaction and looping-condensation. E. Fis residue Asn 73 plays a key role in DNA bending-compaction. Naked λ-DNA (open circles) was incubated with 5 μM Fis mutant N73S; the resulting N73S-DNA complex (filled circles) was “anti-compacted” at high forces (shift to right), indicating that N73S stiffens DNA; looping-collapse at low forces occurred. In a separate experiment with 5 μM N73A (filled squares) a similar anti-compaction effect was observed, along with an incomplete looping-condensation reaction at very low force (below 0.1 pN). F. Crosslinking of Fis dimers does not affect looping-condensation activity. Naked DNA (open circles) was incubated in separate experiments with 5 μM V58C in the presence of diamide to maintain disulfide crosslinks or in the presence of DTT (unlinked). In both cases, looping-condensation occurred.
Figure 8
Figure 8
Gel mobility shift assays evaluating nonspecific binding properties of selected Fis mutants. A. Fis mutant R71K behaves indistinguishably from wild-type Fis. B. R71A, which fails to loop, weakly forms discrete complexes at high protein concentration. C. K90A, which fails to loop, is very defective in nonspecific binding. D. N73S, which forms stiffer complexes that promote weak looping, poorly forms discrete complexes at high protein concentrations. E. K36E, which does not change the elastic properties of DNA at 5 μM, forms nearly a full complement of discrete complexes, but its LMC migrates faster than the wild-type LMC. F. G72D, which exhibits normal compaction and looping at 5 μM, is very defective for in nonspecific binding. For each mutant, Fis was added at 2-fold increasing concentrations beginning at 0.125 nM (lane 1) to 256 nM (lane 12). The DNA probe for each experiment was the 149 bp fragment from the yeast actin gene.
Figure 9
Figure 9. Sketches of Fis binding DNA in different concentration regimes
A. Disperse binding (≤10 nM); bends generated by bound Fis dimers (filled circles) are well separated from one another. B. Coated filament (~20 nM): Fis dimers bound adjacent to one another, roughly 21 bp apart, such that the bends introduced into the DNA are largely in phase (see panel F). C. The “LMC” (≥75 nM): Fis dimers cooperatively assemble onto the Fis-coated complex to form an LMC beginning with a density of about 2 dimers per 21 bp; at higher Fis concentrations increasing numbers of Fis dimers are associated with the LMC. D. Fis-looped DNA (≥1 μM): Fis dimers bound in the LMC are able to stabilize DNA crossings. E. Sketch of Fis dimer clusters organizing a supercoiled E. coli chromosome domain. Localized regions of high density Fis binding that are postulated to occur at clusters of high affinity Fis binding sites within the chromosome. Two remote clusters associate to stabilize a loop containing the intervening DNA. Fis and other DNA binding/bending proteins (grey circles) bound within the intervening DNA will further compact the DNA. F. Structural model of 3 tandem Fis dimers within a coated complex. Each Fis dimer is rendered as yellow and blue subunits and are positioned to account for the DNA cleavages (green nucleotides) generated by Fis98-OP chimeras. Where visible, acetamido-1,10-phenanthroline are red sticks with copper ions as orange spheres.

Similar articles

See all similar articles

Cited by 65 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources