Fig 1.
Binding of ATP-DnaA-his to genomic DNA in vitro.
The relative amount of binding by ATP-DnaA-his is plotted on the y-axis (normalized so that maximum binding has an amplitude of 1) versus the position along the chromosome on the x-axis. The amount of binding was determined by sequence analysis of the DNA recovered in each binding reaction. Binding data is presented in 200 nucleotide bins, with the maximum binding amplitude in each bin drawn. The 4.2 mb circular chromosome is depicted linearly such that the origin of replication is near the middle of the x-axis. The concentration of ATP-DnaA-his in each binding reaction was (A) no DnaA; (B) 55 nM; (C) 140 nM; (D) 550 nM; (E) 1.4 μM; (F) 4.1 μM. The major peaks are numbered (C), and correspond to the following nearby loci: (1) sda; (2) ywlC; (3) ywcI; (4) yydA; (5) consists of 3 adjacent peaks (trmE, dnaA, and between dnaA and dnaN) that are not resolved at this scale; (6) gcp/ydiF. The inset in panel B above the asterisk corresponds to a 7 kb region that contains the trmE, dnaA, and dnaA/N binding regions.
Fig 2.
IDAP-Seq data for individual regions bound by DnaA.
The 10 strongest binding regions (A-J), plus one weaker binding region (K, L), are displayed, using binding data at 1.4 μM ATP-DnaA-his. The bottom section of each panel shows the genomic coordinates (using the AG1839 genomic sequence), the positions of each gene (gray rectangles), and the gene names. Arrowheads indicate the direction of transcription. The middle section of each panel is a histogram of the number of sequence reads that start at each nucleotide (blue, above the line, for sequence reads mapping to the top strand; green, below the line, for sequence reads mapping to the bottom strand). The red circles indicate DnaA boxes, predicted using the PSSM described in this paper. The top section of each panel is a plot of the amount of DNA recovered (as inferred from the sequence data) versus genome position, using 1.4 μM ATP-DnaA-his (black line) or no DnaA (red lines). The amount of DNA recovered is scaled to a global maximum of 1, as described for Fig 1. A-J. The 10 strongest binding regions, corresponding to peaks #1–10 in Table 1, S1 Table, and S1 Fig. (A) upstream of dnaA (part of oriC); (B) between dnaA and dnaN, containing the DNA unwinding element (also part of oriC); (C) upstream of ywcI and vpr; (D) downstream of gcp and ydiF; (E) upstream of trmE and downstream of jag; (F) upstream of ywlC and downstream of ywlB; (G) upstream of yqeG and sda; (H) upstream of yydA, spanning yyzF, and upstream of yycS; (I) within codV; (J) within rplB. K-L. A representative weaker binding region, (peak #49 in S1 Table; S1 Fig). (K) binding scaled to 1 to be comparable to previous panels; (L) the same region rescaled so that the binding pattern is visible.
Fig 3.
Identification of DnaA binding sites at single nucleotide resolution.
Histograms of the number of sequence reads that start at each nucleotide are plotted as in Fig 2 (blue, above the line, for sequence reads mapping to the top strand; green, below the line, for sequence reads mapping to the bottom strand). Data are from a binding reaction containing 4.1 μM ATP-DnaA-his, except for panel F, where data are from a reaction containing 55 nM ATP-DnaA-his. For each panel, a 300 bp portion of the genome is presented, and the y-axis is scaled so that the data fills the space. Predicted DnaA boxes are represented by red ovals, and are numbered from left to right for regions with more than three DnaA boxes. The gray rectangles below the histograms depict nearby genes, with arrowheads indicating the direction of transcription. The regions presented are: (A) inside cssS; (B) inside ygaN; (C) inside ypiF; (D) inside lpdV; (E) inside yabS; (F) upstream of sda (55 nM ATP-DnaA-his data); (G) upstream of sda (4.1 μM ATP-DnaA-his data). Panel B corresponds to peak #140 (S1 Table and S1 Fig) and panels F and G correspond to peak #7. The other regions did not show sufficient binding at 1.4 μM DnaA to be included in the peak catalog.
Table 1.
Top 20 DnaA binding regions in vitro.
Fig 4.
Comparison of DnaA boxes from a consensus sequence with DnaA boxes from the PSSM.
(A) A logo, drawn using WebLogo [32], of the DnaA boxes used to construct the DnaA box PSSM is shown. (B-E) Histograms of the number of sequence reads that start at each nucleotide are plotted (blue for sequence reads mapping to the top strand; green for sequence reads mapping to the bottom strand). Data are from the binding reaction containing 4.1 μM ATP-DnaA-his. For each panel, a 300 bp portion of the genome is presented, and the y-axes are scaled so that the data fills the space. The red ovals on the horizontal axis indicate the position of DnaA binding sites predicted using the PSSM described here. The pink ovals above the horizontal axis indicate DnaA boxes with 2 mismatches from the TTATNCACA consensus sequence, and the maroon ovals have 1 mismatch from the TTATNCACA consensus. (No perfect matches to the consensus are found in these regions.) The gray rectangles below the histograms indicate nearby genes, with arrowheads indicating the direction of transcription.
Fig 5.
Binding curves for ATP-DnaA-his for selected chromosomal regions.
The relative amount of DNA recovered from different chromosomal regions is plotted on the y-axis, versus the ATP-DnaA-his concentration on the x-axis. The curves were obtained from fitting the data to the Hill Equation. Chromosomal locations (nucleotide position in the sequence of AG1839, peak number in S1 Table, and nearby genes) include: (A) 150, #1, upstream of rpmH and dnaA; (B) 1841, #2, downstream of dnaA and upstream of the DUE and dnaN; (C) 3885674, #5, upstream of ywcI and vpr; (D) 627955, #3, downstream of gcp and ydiF; (E) 4191071, #4, upstream of trmE and downstream of jag; (F) 3772105, #6, upstream of ywlC and downstream of ywlB; (G) 2620059, #7, upstream of yqeG and sda; (H) 4113012, #8, upstream of yydA and downstream of yyzF; (I) 1670814, #9, inside codV; (J) 137727, #10, inside rplB; (K) 624773, #40, inside thiL; (L) 1922157, #127, upstream of ynfC and alsT.
Fig 6.
Relative DNA binding by ATP-DnaA-his compared to ADP-DnaA-his.
(A) The ratio of the amount of DNA recovered bound to ATP-DnaA-his vs. ADP-DnaA-his is plotted versus the DnaA concentration. Background binding was subtracted prior to calculating the ratio. All 269 peaks detected at 1.4 μM were analyzed at each concentration, but the ratio of ATP/ADP binding is shown only if the binding was 1.5-times greater than background for both the ATP and the ADP binding reactions at that DnaA-his concentration. For many weaker peaks, these criteria were met only at the highest concentration of DnaA-his. Solid and dotted lines connect points for the indicated region across concentrations. The black bars indicate the average ratio (ATP-DnaA-his/ADP-DnaA-his) at each DnaA-his concentration. The two data points at 1.4 μM DnaA-his that are slightly less than 1 correspond to the yydA and dnaN regions. (B-G) The relative amount of DNA recovered from different chromosomal regions is plotted on the y-axis, versus the DnaA-his concentration (log scale) on the x-axis. ATP-DnaA-his, open circles and dashed lines (data are the same as shown in parts of Fig 5); ADP-DnaA-his, filled triangles and dotted lines. Genomic coordinates (AG1839 genome), and nearby genes: (B) 2620051, upstream of sda and yqeG; (C) 627955, downstream of gcp and ydiF; (D) 972751, inside yhcN; (E) 1006129, inside yhdF; (F) 150, oriC upstream of dnaA; (G) 1841, oriC upstream of the DUE and dnaN.
Table 2.
Effects of Rok on binding by DnaA in vivo1.