Best ideal: sequence of every from the putative DnaA boxes identified by the PSSM and shown in the middle left. For regions with >5 putative DnaA boxes, the total list is in S5 Table). Bottom suitable: binding curves plotting the volume of DNA recovered as a function in the concentration of DnaA-his. ATP-DnaA-his, open circles and dashed lines; Title Loaded From File ADP-DnaA-his, filled triangles and dotted lines. (PDF) S2 Fig. The C-terminal DNA binding domain of DnaA is required for association of DnaA with chromosomal regions in vitro. Binding reactions were performed beneath precisely the same conditions as for DnaA-his, except that 4.1 M DnaAC-his was made use of. DnaAC lacks the C-terminal 91 amino acids which are needed for DNA binding. A reaction containing full-length DnaA-his was performed in parallel. The binding reactions contained two.5 mM ATP. The recovered DNA was assayed applying qPCR, with the primers indicated in S5 Table. The following loci were assayed (peak numbers refer to those in S1 Fig and S1 Table): cotH (peak 198), ypfD (peak 235), yphF (too weak to become named as a peak at 1.four M DnaA but clearly discernible at four.1 M), ydiO (peak 250), rplB (peak ten), dnaA (peak 1), and nicK, a handle region that does not bind DnaA. (TIFF) S3 Fig. Technique for quantitating binding data over a array of DnaA-his concentrations. In panels A-C, a schematic representation employing a toy dataset shows how deep sequencing data have been converted to coverage along the chromosome. (A) start positions of sequence reads are plotted as histograms, and are shown clustered around a DnaA binding web page depicted by the red dotted line. (B) Each read was extended in the acceptable direction (rightward for reads corresponding for the top rated strand, and leftward for reads corresponding to the bottom strand) by the average fragment length of 250 bp. (C) The amount of fragments containing each nucleotide along the genome is determined, yielding the relative coverage along the genome. Though this allows for comparison among distinctive genomic loci within the similar binding reaction, it does not support comparison in between different binding reactions (i.e., comparing ATP and ADP, or comparing diverse concentrations of DnaA-his.) (D) Actual sequence data from the sda promoter area from samples containing the indicated concentrations of ATP-DnaA-his. The y-axis scale for every on the samples is definitely the exact same. The identical total quantity of reads was mapped for each and every binding reaction, but the variety of reads mapping towards the sda promoter area (and other high-affinity DnaA binding regions) decreased at the two highest concentrations of DnaA-his. This can be for the reason that at these DnaA concentrations, binding to sda has currently saturated, whilst an increasing portion from the reads map to weaker binding regions, and there is also an increase in background binding. (E) The relative coverage along the exact same region as in D, obtained by extending the reads by the average study length and summing the number of extended reads spanning each position, as depicted inside a, B, and C. (F) The amount of DNA recovered in each binding reaction (prior to any preparation measures for deep sequencing) was determined. (G).