Prediction and Validation of Sigma70 Promoters Figure 2. Flow chart of promoter validation. Genome-wide motif search using MEME and MAST provided 3874 and 14962 predicted s70- promoters at p-value cut-offs of 1024 and (1024#p-value,1023), respectively. For validation of the predicted promoters, transcriptionally active regions (TARs) were detected using tiling array data of L. plantarum WCFS1 and for each TAR the transcription start site (TSS) was determined. After filtering the TSSs for low-expressed TARs, 621 TSSs were obtained for validation of predicted s70-promoters. At a low p-value cut-off (p-value,1024), 568 s70-promoters were found that co-localized within a distance of 40 nt of TSSs. An additional 120 s70-promoters were found to co-localized within a distance of 40 nt of TSSs after raising the p-value cut-off (1024#p-value,1023). For 94 TSSs, no co-localized s70-promoter (within a distance of 40 nt) was found. doi:10.1371/journal.pone.0045097.g002 and their relative distance to predicted promoters can be used to corroborate the involvement of these promoters in the initiation of transcription of the corresponding TAR. The TSS locations were correlated to appropriately oriented predicted s70-promoters (p- value,1024) in close proximity of the TSS, identifying 568 s70- promoters within a distance 40 nt of a TSS (see Figure 2) of which 474 s70-promoters were within a distance 20 nt of a TSS (tiling- array resolution 14 nt) (see Figure 3). About 70% of TSSs assigned to highly expressed TARs (group A and B in Figure 4) were in close proximity of a predicted s70-promoter (#40 nt), while for ‘only’ 17% of TSSs detected for low expressed TARs (group E in Figure 4) a s70-promoter could be assigned. Expectedly, TSSs inferred from expression signals of highly expressed genes give better corroboration results than TSSs inferred from expression signals of lower expressed genes. The lower promoter assignment to the low-expression TARs may be due to the ambiguity of the TSS prediction for these TARs, while the higher accuracy of TSS prediction for high expression TARs provides more confident co- PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e45097