Bernard et al. BMC Genomics 2010, 11:166 http://www.biomedcentral.com/1471-2164/11/166 Page 3 of 15 Figure 1 Distribution of TATAWA in plant promoters aligned relative to the TSS. The canonical TATA-box defines the TATAWA- PLM. The distribution model is learned in the [-1000, -300] region where the base line (continuous line) and the upper bound of the confidence interval (dashed line) are estimated and applied to the [-300, 500] region. Distributions are shown for the Arabidopsis thaliana set containing 14927 promoters (A) and for the Oryza sativa set containing 18012 promoters (B). The TATAWA-PLM is preferentially positioned 32 bases upstream of the TSS with a [-39, -26] functional window in both genomes. A. thaliana and O. sativa are characterized by an SMS of 21 and 19 respectively. expected position [31,32]. We observed PLMs with the same positional constraints as TATAWA, i.e. showing a sharp peak in the [-39, -26] region. These PLMs may be (i) functional motifs hereafter named as TATAΔ-PLMs where Δ stands for variant; or (ii) motifs being PLMs due to their overlap with the TATAWA sequence. This overlap represents an inherent drawback of the method since motifs shorter than 6 nucleotides (TAT for instance) as well as 6 base long motifs (NNTATA for instance) may appear as PLMs. TATA-variant and TATA-box are distinct regulatory elements that need differentiating [33]. For this reason, we searched for TATAΔ-PLMs in sets of promoters not containing the canonical TATA-box in the [-39, -26] region. In this way, we were able to ascertain that the PLMs found within the promoter sets were bona fide elements under topological constraint. In A. thaliana and O. sativa respectively 12321 (82.5%) and 15411 (85.6%) promoters were found without a TATAWA in the [-39, -26] region. Relative to the TATAWA sequence, 32 possible motifs diverged at one position. Out of these 32 motifs, only 11 were found to represent PLMs (TATAΔ1- PLMs) conserved in both species (Figure 2, column 2). Consistent with in vivo experimental results [34], T to A Figure 2 Sequence distance graph of TA-PLMs present in A. thaliana promoters. The TATA-box PLM sequence (first column), TATAΔ1-PLM sequences (second column) and TATAΔ2-PLM sequences (third column) are organized in an oriented graph. In red the motifs observed in more than 350 promoters; in orange the motifs observed in more than 200 promoters and in black the motifs observed in up to 200 sequences. Each edge between two PLMs reflects the presence of one substitution leading from one PLM to another one. Numbers of A. thaliana promoters containing a given motif are indicated above the sequences. The TATAWA motifs are seeds (in bold). or A to T substitutions were the main differences observed among the TATAΔ1-PLMs. Motifs with a base C or G substitution at the central positions were not considered as PLMs and were counter-selected. Motifs with a C or G substitution at the T1 or A6 positions were, however, considered as TATAΔ1-PLMs. There remained 9512 (63.7%) and 13494 (74.9%) of A. thaliana and O. sativa genes respectively containing neither a TATAWA nor a TATAΔ1-PLM. We identified only 4 conserved TATAΔ2-PLMs amongst the 211 possible motifs (Figure 2, column 3) and no conserved TATAΔ3-PLM. Finally, at the TATA-box expected posi- tion, 4151 A. thaliana (27.8%) and 3045 O. sativa pro- moters (16.9%) contained a TATAΔ-PLM. Hereafter, TATAΔ- and TATAWA-PLMs are collectively referred to as TA-PLMs.