916 Nucleic Acids Research, 2011, Vol. 39, No. 3 Figure 1. Puriﬁcation of the B. subtilis sA with a core-independent promoter DNA-binding activity. (A and C) Puriﬁcation of the in vitro refolded B. substilis sA by molecular sieving columns, Superdex HR-200 (A) and SD-75 (C). AU as shown in the left panel of (A) indicates the absorption unit of the fractionated samples. The fraction no. shown on top of the SDS–polyacrylamide gels indicates the fraction of the sA-containing sample collected from the two columns. (B and D) The G3b promoter DNA-binding activity of sA fractionated by Superdex HR-200 (B) and SD-75 (D) columns. The numbers above the horizontal lines are the fractions of sA-containing sample collected from the two columns. The numbers shown below the horizontal lines are the concentrations (mM) of sA used. methods used to purify the overexpressed sA from E. coli (16,47,48). Basically, the sA-containing inclusion bodies were denatured with 7 M Gn–HCl and diluted slowly with refolding buffer drop by drop, rather than stepwise. Then, the refolded sA was subjected to puriﬁcation with two consecutive molecular sieving columns, Superdex HR-200 and SD-75, rather than with an anion exchange (DEAE cellulose) and a molecular sieving (Sephadex-G75) column. With these modiﬁcations, the sA with promoter DNA-binding activity was achieved. Shown in Figure 1A and B are the results of sA puriﬁcation with Superdex HR-200 and the promoter DNA-binding activities of the fractionated sA as examined by EMSA. Signiﬁcant binding of the tested f29 phage G3b promoter DNA was observed for the sA eluted earlier from the Superdex HR-200 column (fraction nos 14–16), suggesting that the sA active in promoter DNA binding has a rela- tively looser conformation. Because the sA sample was still low in homogeneity at this step, we further puriﬁed it with an SD-75 molecular sieving column. As shown in Figure 1C and D, the sA eluted from the SD-75 column (fraction nos 21–23) had a much improved homogeneity ( 95%) and was also active in promoter DNA binding with a relatively high apparent dissociation constant (Kd) of about 5 mM, which is similar to that reported for the complex of B. subtilis sD and its cognate promoter (17). Because the active sA sample is free from the b-subunit of E. coli core RNAP and lack of transcription activity A (Supplementary Figure S1), we assumed that the s , by itself, is able to bind the G3b promoter DNA. To rule out the possibility that the promoter DNA-binding activity of the in vitro refolded sA is just an artifact due to protein refolding, we further puriﬁed the sA overproduced in E. coli in a soluble form at low temperature (16 C) and analyzed its promoter A DNA-binding activity. Similar to the refolded s , the soluble sA was able to bind the G3b promoter DNA in- dependent of core RNAP (Supplementary Figure S2A and B), supporting the hypothesis that the B. subtilis sA is an intrinsic DNA-binding protein. pA binds preferentially to the promoter element in vitro 10 To investigate whether sA has preference for a speciﬁc promoter element, six Mu G3b promoter DNA with single or pairwise substitutions at 10 element, 35 element and TG motif (Figure 2A) were adopted for competition assay by EMSA. In this analysis, the binary complex preformed by sA and labeled Wt G3b promoter DNA was challenged with various concentra- tions of the unlabeled Wt, Mu G3b promoter DNA and non-promoter DNA (the full-length tgbp2 cDNA of Bamboo mosaic virus). As shown in Figure 2B, the pre- formed binary complex was relatively more sensitive to challenge with the Wt or the Mu G3b promoter DNA having base substitutions at either 35, TG or 35/TG element than to challenge with those having substitutions at 10 element (including 10, 10/TG and 10/-35) or with the non-promoter DNA. The requirement of the promoter 10 element for efﬁcient competition was also visualized when the correlation between the fraction- al retention of the preformed sA-promoter DNA complex and the log [Competitor]/[Labeled G3b promoter DNA] was examined (Figure 2C). Clearly, sA binds preferentially to the 10 element of G3b promoter in vitro. Moreover, the high efﬁciency of the non-promoter DNA, albeit lower than that of the Wt