914 Nucleic Acids Research, 2011, Vol. 39, No. 3 Besides the recognition of 10 element, TG motif and 35 element by s, the spacer DNA, which separates the 10 and 35 elements and is important for a promoter to function, is also proposed to be recognized by s (30). In general, it is the length of the spacer DNA (spacing), rather than the sequence (except the TG motif), which is highly homologous (31–33). Usually, the 17 ± 1 bp spacing confers the optimal activity on promoters recognized by the primary s (34–39). The main roles of the spacer DNA are thought to hold a more favorable orientation of the 10 element relative to the 35 element for initial binding of RNAP and for facilitating subsequent formation of a competent RNAP–DNA open complex (40–46). However, the mechanism leading to the recognition of optimal promoter spacing by RNAP remains unclear. Using a series of glutathione S-transfer- ase (GST)-fused partial s70 polypeptides, which exhibit speciﬁc binding to either one or both of the 10 and 35 elements of tac promoter, for competition assay, it was inferred that the s70, by itself is a molecular ruler, which is able to discern the optimal spacing of the tac promoter (23,30). In this study, we also re-examined the A inference using the B. subtilis s . Here, we provide in vitro evidence demonstrating that the house-keeping B. subtilis sA is able to bind speciﬁcally to the 10 element of promoters independent of core RNAP. However, the sA is unable to discern the optimal promoter spacing. To fulﬁll this goal, it requires the assistance from core RNAP. MATERIALS AND METHODS Overproduction and puriﬁcation of pA Methods used for overproduction and puriﬁcation of sA in inclusion bodies are similar to those reported previously (47). The cell lysate was centrifuged at 7500g to harvest the sA-containing inclusion bodies that were then denatured with TEDG buffer [10 mM tris–hydrochloric acid (HCl), pH 7.9, 0.1 mM ethylenediaminetetraacetic (EDTA), 0.1 mM dithiothreitol (DTT), 0.2 mM phenyl- methanesulfonylﬂuoride (PMSF), and 5% glycerol] supple- mented with 200 mM NaCl and 7 M guanidine–HCl (Gn–HCl), and refolded through drop-by-drop dilution of the denatured protein with the TEDG buffer containing 200 mM NaCl until the concentration of Gn–HCl was 55 mM. The refolded sA was centrifuged at 7500g to remove unfolded proteins. The soluble sA in the super- natant was then precipitated with 40% (w/v) ammonium sulfate and resuspended in TEDG buffer containing 200 mM NaCl before further puriﬁcation with the molecu- lar sieving columns, Superdex HR-200 and Superdex SD-75. To overproduce soluble sA, Escherichia coli BL21 (DE3)/pCD2 or E. coli BL21 (DE3)/pCD2M, which is able to express the wild-type (Wt) sA or the mutant (Mu) sA with Gln-196-Ala and Thr-199-Ala substitutions, respectively, was grown at 37 C to an OD550 of 0.5 before being transferred to 16 C. After incubation of the culture at 16 C for 10 min, isopropyl b-D-1-thiogalactopyranoside (IPTG) (ﬁnal concentration 0.4 mM) was added to induce the expression of sA. Fifty minutes later, rifampicin (ﬁnal concentration 100 mg/ml) was added into the culture which was further incubated at 16 C for 12 h. The E. coli cells were harvested and disrupted with a microﬂuidizer in TEDG buffer supplemented with 100 mM NaCl and then centrifuged at 7500g. The soluble sA in the super- natant was absorbed with 20 g of diethylaminoethyl cellu- lose (DEAE)-cellulose pre-equilibrated with TEDG buffer containing 100 mM NaCl. The DEAE cellulose was packed into a column and a NaCl gradient from 0.1 to 0.45 M was applied. The major sA-containing fractions were pooled and the sA were precipitated with 40% (w/v) ammonium sulfate and resuspended in TEDG buffer con- taining 200 mM NaCl before further puriﬁcation with the molecular sieving columns, Superdex HR-200 and Superdex SD-75. Both the refolded and soluble sA puriﬁed had homo- geneities higher than 95% and were stored in storage buffer (20 mM Tris–HCl, pH 7.9, 10 mM MgCl2, 1 mM EDTA, 200 mM KCl, 0.1 mM DTT, 0.2 mM PMSF and 50% glycerol). Mutagenesis of the Gln-196 and Thr-199 in the Region 2.4 of pA The sigA gene with Gln-196-Ala and Thr-199-Ala substi- tutions was constructed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The plasmid, pCD2, which contains the full-length sigA gene, was used as the DNA template for mutagenesis. The two primers used for codon replacement are as follows: sigA-196- 199A-F: 50-GCTACGTGGTGGATCAGAGCGGCGAT TGCACGCGCCATTGCCGATC-30 and sigA-196-199A- R: 50-GATCGGCAATGGCGCGTGCAATCGCCGCT CTGATCCACCACGTAGC-30. The plasmid containing the correct mutant sigA gene was named pCD2M. Construction of the spacing variants of the B. subtilis trnS promoter DNA The trnS spacing variants with a 14- to 21-bp spacing (Figure 6A) were created by the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The templates and primer pairs used for the construction were shown in Supplementary Table S1. All of the resultant spacing variants were sequenced to be correct and were named as trnS-X, in which X indicates the length of spacer ranging from 14 to 21 bp. Construction of the Mu G3b and trnS-17 promoters used for competition assay The mutation at the 10, 35 element or TG motif of G3b or trnS-17 promoter was created using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The templates and primer pairs used for the construction were shown in Supplementary Table S1. All of the promoter mutations were conﬁrmed by DNA sequencing. To prepare competitor DNA, the Wt and Mu G3b promoter DNA fragments (182 bp in length, spanning from +67 to 115 of the G3b promoter) were synthesized by polymerase chain reaction (PCR). The Wt G3b promoter DNA-containing plasmid, pCT-G3b and the