pCT-G3b-derived plasmids containing mutation at 10, 35 element, TG motif or either two elements of the G3b promoter were used as templates and BC1041-BamHI (50-GAGCTCGGATCCAGAGAACGTAGACAACA A-30) and BC1046-HindIII (50-CTGCAGAAGCTTGCC ATTTCTTCGTCCCACTTTC-30) as primers. Similarly, the Wt and Mu trnS-17 promoter DNA fragments (95 bp in length, spanning from +15 to 80 of the trnS- 17 promoter) were synthesized by PCR. The Wt trnS promoter DNA-containing plasmid, pOtrnS and the pOtrnS-derived plasmids containing mutation at either 10, 35 or both elements of the trnS-17 promoter were used as templates, and trnSF5 (50-CATTATAAAAGTCG TCACG-30) and trnSR3 (50-TGTGAATAATGAGACAA GAC-30) were used as primers. The promoter DNA frag- ments thus synthesized were further run on a 2% agarose gel, cut out from the gel, and puriﬁed using the Micro-Elute DNA Clean/Extraction Kit (GeneMark). The puriﬁed DNA was diluted with DNA-binding buffer [50 mM Tris–HCl, pH 7.9, 10 mM MgCl2, 160 mM KCl, 0.1 mM EDTA, 0.1 mM DTT and 5% (v/v) glycerol] before being used. Synthesis and labeling of the promoter DNA fragments The Wt and Mu G3b promoter DNA were synthesized by PCR using the pCT-G3b or the pCT-G3b-derived plasmids containing mutation at 10, 35 or both elements of the G3b promoter as DNA templates and BC1041-BamHI and BC1046-HindIII as primers. The promoter DNA frag- ments of trnS-X were synthesized by PCR using the pOtrnS (containing trnS-17) and pOtrnS-derived plasmids containing trnS-X (X is from 14 to 21 except 17) as templates and trnSF5 and trnSR3 as primers. To label the 50-end of promoter DNA fragments, either reverse or forward primer was labeled by [g-32P]-adenosine triphos- phate (ATP) prior to PCR. For labeling template strand DNA, reverse primer (BC1046-HindIII or trnSR3) was 32P–labeled; for labeling non-template strand DNA, forward primer (BC1041-BamHI or trnSF5) was 32P– labeled. To label primer, 20 pmol of the primer DNA was mixed with 20 pmol of [g-32P]-ATP and 10 U of T4 poly- nucleotide kinase (Roche) in 20 ml of phosphorylation buffer (50 mM Tris–HCl, pH 8.2, 10 mM MgCl2, 0.1 mM EDTA, 5 mM DTT and 0.1 mM spermidine), then the mixture was incubated at 37 C for 2 h. The labeled promoter DNA fragments were run on a 2% agarose gel, cut out from the gel and puriﬁed using the Micro-Elute DNA Clean /Extraction Kit (GeneMark). The puriﬁed DNA was diluted with DNA-binding buffer before being used. Electrophoretic mobility shift assay For electrophoretic mobility shift assay (EMSA), the sA or sA-RNAP was incubated with 32P-labeled G3b (1 nM) or trnS (0.5 nM) promoter DNA at 37 C for 10 min. The ﬁnal volume of the binding mixture was adjusted to 10 ml by adding the DNA-binding buffer. The binding of sA to promoter DNA was performed in the presence of 0.1 mM heparin. The complex of sA and promoter DNA was then run on a 5% non-denaturing polyacrylamide gel in 1 Nucleic Acids Research, 2011, Vol. 39, No. 3 915 tris–acetate–EDTA (TAE) buffer (40 mM tris–acetate, A pH 8.5 and 2 mM EDTA) at 4 C. The complex of s - RNAP and promoter DNA was run at room temperature. Finally, the gel was dried and analyzed with a Fuji BAS 2500-phosphoimager. For reconstitution of holo RNAP, core RNAP and 10-fold molar excess of sA were incubated at 37 C for 10 min. A In EMSA-based competition assay, the preformed s - G3b promoter DNA complex obtained by incubating 32P-labeled G3b promoter DNA (1 nM) with an amount of sA in the presence of 0.1 mM heparin at 37 C for 10 min was challenged with the cold Wt, Mu G3b promoter DNA or non-promoter DNA (tgbp2 cDNA of Bamboo mosaic virus) for another 10 min. Then, the samples were run on a 5% non-denaturing polyacrylamide gel in a 4 C-cold chamber. Similarly, the sA-trnS-X promoter DNA complex was preformed by mixing 32P-labeled trnS-X promoter (0.5 nM) with sA in the presence of 0.1 mM heparin. Then, the cold Wt or Mu trnS-17 promoter DNA was added. The mixture was further incubated at 37 C for 10 min before being electrophoresed at 4 C. The fractional retention of the preformed binary complex was measured as a function of the molar ratio of competitor DNA to labeled promoter DNA. DNase I footprinting assay The sA or sA-RNAP was incubated with 0.5 nM 32P-labeled G3b or trnS promoter DNA for 30 min at various temperatures (20, 25 and 37 C). The ﬁnal volume of the binding mixture was 40 ml and the ﬁnal concentrations of buffer components in the binding mixture were 41.6 mM Tris–HCl, pH 7.9, 10 mM MgCl2, 0.1 mM DTT, 0.35 mM EDTA, 60 mM KCl, 100 mg/ml bovine serum albumin (BSA) and 17.6% glycerol. After incubation, an appropri- ate amount of DNase I (Roche) was added to digest the DNA. For binding at 20 C, 0.6 (for G3b promoter) or 0.25 U (for trnS promoter) of DNase I was added; for binding at 25 C, 0.4 U (for G3b promoter) of DNase I was added; for binding at 37 C, 0.25 (for G3b promoter) or 0.1 U (for trnS promoter) of DNase I was added. After a proper period of time, 40 ml of stop buffer (50 mM Tris– HCl, pH 7.9, 200 mM NaCl, 20 mM EDTA and 1 mg/ml glycogen) was added to stop the digestion reaction. The digested DNA was extracted with phenol–chloroform. The DNA fragments in the upper aqueous layer were col- lected and precipitated with 0.3 M NaOAc and 2.5-fold volumes of 100% ethanol, washed twice with 70% ethanol, heat dried (60 C), dissolved in 10 ml of loading buffer (98% deionized formamide, 10 mM EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol) and electrophoresed with an 8% (w/v) polyacrylamide/8 M urea sequencing gel in 1 tris–borate–EDTA (TBE) buffer (90 mM tris–boric acid, pH 8.5 and 2 mM EDTA). RESULTS Puriﬁcation of the B. subtilis primary p, pA, with a core-independent promoter DNA-binding activity The B. subtilis sA is a primary s factor. To obtain the sA active in promoter DNA binding, we modiﬁed the