922 Nucleic Acids Research, 2011, Vol. 39, No. 3 A Shown in Figure 8B are the footprinting patterns of s - RNAP on both DNA strands of the trnS spacing variants at 37 C. Again, signiﬁcant footprints of sA-RNAP at both 10 and 35 elements, consistent with the existence Figure 7. sA contacts the trnS spacing variants in a ﬂexible manner in addition to the 10 speciﬁc interaction. The footprinting patterns of sA on the template (left panel) and non-template strand DNA (right panel) of the trnS spacing variants at 20 C. In this assay, 20 mM sA was used for footprinting assay. The numbers above the horizontal lines are the spacer lengths of the trnS spacing variants. The plus and minus signs A denote the addition and omission of s , respectively. The numbers shown on the left of each panel are positions relative to the transcrip- tion start site of the trnS-17 promoter. The DNA protected by sA on both the template and non-template strand DNA of each spacing variant are encompassed by the upward and downward arrowheads. The promoter 10 and 35 elements protected by sA on each trnS spacing variant are indicated by dotted lines. of speciﬁc interaction between sA-RNAP and the two promoter elements (Supplementary Figure S6), were observed only for the trnS variants with an optimal spacing, especially that with a 17-bp spacer. No or very weak footprint of sA-RNAP was detected on both DNA strands if the spacer is shorter (14- or 15-bp) or longer (19- , 20- or 21-bp). Taken together, these results demonstrated that core RNAP not only enables sA to discern the optimal promoter spacing but also to simultaneously and speciﬁcally recognize both promoter elements as previously reported (27,29). DISCUSSION We have documented the in vitro core-independent promoter-speciﬁc interaction of the B. subtilis primary s factor, sA. This interaction is 10 speciﬁc; however, it is unable to allow the sA to discern the optimal promoter spacing. To fulﬁll this goal, the sA requires assistance from core RNAP. The detection of core-independent 10 speciﬁc pro- moter interaction of sA in vitro is contrary to previous ﬁndings with the E. coli s70 (22,23) and T. maritima sA (24,25), for which it has been reported that Region 1.1 of s acts as an auto-inhibitor for s-promoter interaction, and that the promoter interaction in the absence of core RNAP would require deletion of Region 1.1 of s, just as seen for the sD of B. subtilis (17). Reasons responsible for these contradictory results are complicated. We thought that our ability to detect the core-independent 10 speciﬁc promoter interaction of sA is ascribed to the isolation of sA with a proper promoter DNA-binding conformation through the use of a molecular sieving column, Superdex HR-200 (Figure 1) and the use of a relatively low tempera- ture and low ionic strength for DNase I footprinting Figure 8. The footprinting patterns of trnS spacing variants in response to sA-RNAP binding. (A and B) The footprinting patterns of sA-RNAP on the trnS spacing variants at 20 (A) or 37 C (B). The concentration of sA-RNAP used for footprinting assay was 15 nM. Details for labeling the ﬁgures are the same as those described in the legend to Figure 7.