920 Nucleic Acids Research, 2011, Vol. 39, No. 3 Figure 5. The effect of Gln-196-Ala and Thr-199-Ala substitutions in Region 2.4 of sA on the promoter DNA-binding activity of sA. The sA with Gln-196-Ala and Thr-199-Ala substitutions was designated as Mu-sA. (A) The effect of Gln-196-Ala and Thr-199-Ala substitutions in Region 2.4 of sA on the promoter DNA-binding activity of sA as examined by EMSA. (B) The effect of Gln-196-Ala and Thr-199-Ala substitutions in Region 2.4 of sA on the promoter DNA-binding activity of sA as examined by DNase I footprinting. In both (A) and (B), the numbers shown under Wt-sA or Mu-sA are the concentrations (mM) of s A . Other details for labeling of the ﬁgures are the same as those described in the legend to Figure 3. (C) The effect of Gln-196-Ala and Thr-199-Ala substitutions in Region 2.4 of sA on the preferential promoter 10 binding of sA. In the analysis of fractional retention of the Wt- or Mu-sA-promoter DNA complex as a function of increasing molar ratio of competitor to 32P-labeled G3b promoter DNA, 8 mM Wt-sA or 15 mM Mu-sA was mixed with 1 nM 32P-labeled G3b promoter DNA to generate the sA-promoter DNA complex. For competition, the molar ratios of the competitor DNA to the labeled G3b promoter DNA were 0:1, 0.1:1, 0.5:1, 1:1, 5:1, 10:1, 50:1 and 100:1. The band density of each preformed binary complex in the absence of competitor DNA was referred to as 100%. The experiment was repeated twice and the same tendency was obtained. To start with, we screened the optimal sA concentration for speciﬁc interaction between sA and trnS-17 promoter at 20 C. Speciﬁc footprints of sA on both DNA strands of the trnS-17 promoter, spanning from about 58 to 6 on the template and from about 59 to +3 on the non-template strand DNA (as encompassed by the upward and downward arrowheads), were detected when 20 mM of sA was used for binding analysis (Supplementary Figure S5). Thus, the same condition was adopted for DNase I footprinting analyses of sA on other trnS spacing variants. As shown in Figure 7, similar protection patterns, except those in the spacers, were observed for sA on the trnS spacing variants. Moreover, clear footprint was observed at 10 element on the template strand (left panel), but at both 10 and 35 elements on the non-template strand DNA (right panel). Furthermore, there was a gradual upstream extension of the sA footprint on both DNA strands (indicated by the arrowhead) as the spacing was increased from 14 to 21 bp. Since sA only binds speciﬁcally to the promoter 10 element of the trnS spacing variants (Figure 6C) and since there is a gradual upstream extension of the sA foot- print on the trnS promoter DNA in response to the increase of spacer length, we favored that, in addition to the 10 speciﬁc promoter interaction, sA also contacts the 35 element and its upstream sequence of each trnS spacing variant in a ﬂexible manner. pA-RNAP, but not pA alone, can recognize optimal promoter spacing To examine the effect of core RNAP on binding of sA to the trnS spacing variants, the footprints of sA-RNAP on the spacing variants were analyzed at both 20 C and 37 C. Shown in Figure 8A are the footprints of sA-RNAP on the