Nucleic Acids Research, 2011, Vol. 39, No. 3 921 Figure 6. The promoter 10 speciﬁc interaction of sA is unable to allow the sA to discern the optimal promoter spacing in vitro. (A) The spacing variants of the B. subtilis trnS promoter used in this study. The highly conserved 10 and 35 elements of the trnS promoter are underlined. (B) The binding of sA (top panel) and sA-RNAP (bottom panel) to the trnS spacing variants. In EMSA, 10 mM sA or 50 nM sA-RNAP was used. The numbers above the horizontal lines are the spacer lengths of the trnS spacing variants. The plus and minus signs denote the addition and omission of sA or sA-RNAP, respectively. The values shown under each panel are the percentages of trnS spacing variant bound by sA or sA-RNAP ([Bound DNA]/[Total DNA] %). Each value was the average of triplicate measurements with a standard deviation (STDEV) shown. (C) Upper panel: the Wt and Mu trnS-17 promoters with base substitutions at either 10, 35 or both elements (indicated by 10, 35 and 10/ 35) used for competition assay. The sequences of conserved 10, 35 elements and TG motif of the trnS-17 promoter are underlined, and base substitutions in each Mu promoter are italicized. Lower panel: the fractional retention of the preformed sA-trnS promoter DNA complex as a function of the molar ratio of competitor DNA to 32P-labeled trnS spacing variant ([Competitor]/[Labeled DNA]). The trnS spacing variant is indicated by trnS-X (X is the length of spacer DNA ranging from 14 to 21 bp) above each chart. In this assay, 6 mM sA and 0.5 nM trnS-X promoter DNA were used to generate the sA- trnS-X promoter DNA complex. The molar ratios of competitor DNA to the labeled trnS promoter DNA were 0:1, 1:1, 2:1, 5:1, 10:1, 25:1, 50:1 and 100:1. The band density of each preformed binary complex in the absence of competitor DNA was referred to as 100%. A trnS spacing variants at 20 C. Signiﬁcant footprints of s - RNAP at the 10 and 35 elements of both DNA strands were detected only for the trnS variants with an optimal spacing (16 and 17 bp). Weak or no footprint of sA-RNAP at the 10 element was observed for the variants with a shorter (14- or 15-bp) or a longer (18-, 19-, 20- or 21-bp) spacer; the footprint on this type of promoter was restricted to the region spanning from the upstream of the 10 element to the position corresponding to 60 of trnS-17 on the template strand DNA and from the 35 element to the position corresponding to 59 of trnS-17 on the non-template strand DNA. Clearly, the sA-RNAP mainly binds the 35 element and its upstream sequence at 20 C if the promoter spacing is non-optimal.