Nucleic Acids Research, 2011, Vol. 39, No. 3 919 Figure 4. The effect of base substitutions on the footprinting patterns of sA on the G3b promoter DNA. The effect of base substitutions on the footprinting patterns of sA and sA-RNAP on the template (left panel) and non-template strand DNA (right panel) at 20 C. Wt, 10, 35 and 10/ 35 indicate the Wt G3b promoter and the Mu G3b promoter containing base substitution at 10, 35 or both elements, respectively. Other details for experimental conditions and labeling of the ﬁgures are the same as those described in the legend to Figure 3. indicated that the promoter interaction of sA in vitro is indeed 10 speciﬁc. The 10 speciﬁc promoter interaction of pA is unable to allow the pA to discern the optimal promoter spacing in vitro To see whether sA, by itself, is able to discern the optimal promoter spacing as reported previously (30), the spacing variants of another B. subtilis promoter, trnS, of which the 10, 35 elements, TG motif, and spacing (17 bp) are highly conserved, were constructed. The resultant spacing variants were named as trnS-X, in which X is the length of spacer DNA ranging from 14 to 21 bp (Figure 6A). Moreover, to diminish the potential non-speciﬁc binding of sA to the spacing variants in both upstream and downstream regions of the promoter, a shorter DNA fragment of each spacing variant, corres- ponding to the region from +15 to 80 of the trnS-17 promoter, was adopted to perform EMSA. The results of EMSA are shown in Figure 6B. sA bound to the spacing variants with about equal efﬁciency; however, three distinct binding strengths of sA-RNAP were observed among the spacing variants. Optimal binding was seen for trnS-17. Moderate binding was detected for trnS-16 and trnS-18. Lowest binding was observed for the rest of trnS spacing variants with a shorter (14- or 15-bp) or a longer (19-, 20- or 21-bp) spacer. Since the footprint of sA on the trnS promoter was detectable at 20 but not 37 C, we thus also carried out the binding analysis at 20 C throughout the EMSA. Similar EMSA results were obtained for both sA and sA-RNAP (Supplementary Figure S4). Therefore, sA, by itself, is unable to discern the optimal promoter spacing (17 ± 1 bp). To accomplish this function, the as- sistance from core RNAP is required. To see whether the 10 speciﬁc interaction of sA also occurs with the trnS spacing variants, three Mu trnS-17 promoters with base substitutions at 10, 35 or 10/ 35 elements were constructed (top panel of Figure 6C) and then used for EMSA-based competition analyses. As shown in the bottom panel of Figure 6C, the preformed binary complex of sA and each spacing variant was rela- tively more sensitive to challenge with the Wt or the Mu trnS-17 promoter containing base substitutions at 35 element than with those bearing base substitutions at 10 element (including 10 and 10/ 35 mutations). Clearly, similar to the 10 speciﬁc G3b promoter inter- action of sA, the sA also has a preference for binding to the 10 element of the trnS spacing variants. These results also indicated that the promoter 10 speciﬁc interaction of sA is unable to allow the sA to discern the optimal promoter spacing in vitro. pA contacts the trnS spacing variants in a ﬂexible manner, in addition to the 10 speciﬁc interaction To conﬁrm that sA binds the trnS spacing variants through preferential interaction with their promoter 10 elements, DNase I footprinting assay was performed.