Prediction and Validation of Sigma70 Promoters Figure 1. Highly conserved motifs. Highly conserved motifs (E-value,10280) detected by MEME search in the upstream regions of protein- encoding genes (for a full list see Figure S1 in Supplementary results). Motifs are shown as Seqlogos [45]. doi:10.1371/journal.pone.0045097.g001 in signal intensity at the 59-end of a TAR and by linear extrapolation of the local transcription signal-intensity slope estimates the position of the TSS of the TAR. We evaluated the performance of our TSS predictions by correlating the tiling array- based TSSs with those that were previously identified in L. plantarum by primer extension. For nearly all cases (11 out of 12 genes) the putative TSS predicted on basis of the tiling array data was within a maximum distance of 28 nt from the experimentally TSS determined by primer extension (see Results S3). The majority of these genes (8 genes) were highly expressed in the tiling array dataset (their average expression signal belonged to the two groups of TARs that showed the highest expression levels, see Method). Application of the algorithm allowed prediction of a TSS for each of the 1167 TARs. Expectedly, out of the 1167 predicted TSSs, the majority were located upstream of protein encoding genes (912). Next to these expected TSSs, 149 of the predicted TSSs appeared to be positioned in an antisense orientation relative to the annotated genes, while an additional 106 predicted TSSs were positioned in genomic regions that lack genetically linked genes, suggesting that these TSSs could represent the start of non- coding RNAs (see Figure 2). Validation of predicted s70 promoters using TSSs Experimentally verified TSSs are commonly located between 8– 14 nucleotides downstream of their cognate promoter 210 sequence element [3]. Therefore, the positions of inferred TSSs PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45097