(Figure 3). The early elution of the sA active in promoter-DNA binding from the Superdex HR-200 column suggests that the sA has a relatively looser con- formation. This idea can be supported by our preliminary observation that a sA preparation more active in promoter DNA binding possesses a higher intensity of tryptophan ﬂuorescence (the two tryptophan residues of the B. subtilis sA are located just at the N-terminal end of the promoter 10-binding helix of sA). Possibly, the close proximity of the negatively charged Region 1.1 to the positively charged promoter recognition domains of sA (25) is at least to a certain extent alleviated during A the slow refolding of the denatured s . Moreover, the need of a low temperature and low ionic strength for the detection of an efﬁcient interaction between sA and promoter DNA by DNase I footprinting suggests that the sA-promoter DNA interaction is an exothermic reaction, in which electrostatic and/or hydrogen bonding occur. A The preferential binding of s , by itself, to the promoter 10 (TATAAT) but not 35 element in vitro (Figures 2B, C and 6C) is consistent with the ﬁndings that the consensus sequences of GTA(C/T)AATGGGA and T GTAGAAT, both of which contain the TATAAT-like sequence, are required for single stranded deoxyribonucle- ic acid (ssDNA) aptamer binding to the Thermus aquaticus free sA (54,55). It is also consistent with our observation that the 10 speciﬁc promoter interaction of sA is unable to allow the sA to discern the optimal promoter spacing (Figure 6). The lack of binding speciﬁcity of the promoter 35 element by sA can be attributed to the presence of A Region 1.1 of s , which makes s Region 4.2 inaccessible and thus prevents the interaction between s Region 4.2 and the promoter 35 element (22–24). The ability of sA to contact the promoter 35 element and its upstream sequence of each trnS spacing variant in a ﬂexible manner (Figure 7) indicates that after the promoter 10 speciﬁc interaction of sA, an extensive sA-promoter contact in the 35 and its upstream region occurs. It also suggests that the structure between the two DNA-binding domains of sA is ﬂexible enough for sA to contact the two promoter regions separated by various spacings. The structural ﬂexibility of E. coli s70 in recognition of the 10- and 35-like sequences has also been reported in the Q-engaged transcription elongation complex (56). The structural ﬂexibility between the two DNA-binding domains of sA can be attributed to the ex- istence of a relatively unstructured linker, Region 3.2, in sA according to the modeled structure of thermophilic eubacteria sA (57,58). In fact, our analysis of the struc- tural property of the B. subtilis sA by PONDR also revealed the existence of a disordered ﬂexible Region 3 in sA (Supplementary Figure S7). Thus, the ability of sA in the context of RNAP to discern the optimal promoter spacing must be due to the change of the linker domain from an extended unfolded conformation to a hairpin loop structure ﬁxed in the binary complex of RNAP and promoter DNA (58). This change makes the distance ( 50 A˚ ) between the two DNA-binding domains of s only more compatible with the 17-bp (57.8 A˚ ) spacing (27,59). Nucleic Acids Research, 2011, Vol. 39, No. 3 923 The low sequence selectivity of sA (Figure 3 and Supplementary Figure S3) together with the in vitro 10 speciﬁc promoter interaction of sA (Figures 2B, C and 6C) suggests s can either directly recognize the promoter 10 element or indirectly through non-speciﬁc interaction with the A-T rich or promoter 10-like sequences ubiquitous on the chromosome DNA, especially in the upstream region of a promoter, and through sliding to the promoter site in a way similar to that proposed for the sliding of E. coli lac repressor to its operator (60,61). Investigation of the existence of in vivo sA-promoter inter- action, which is critical to s function, is currently under- going in the lab. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank the National Science Council of Taiwan, Republic of China, for supporting the research. FUNDING National Science Council of Taiwan, Republic of China (NSC 92-2311-B-005-021, NSC 94-2311-B-005-006, and NSC 94-2311-2752-B-005-001-PAE). Funding for open access charge: National Science Council of Taiwan, Republic of China. Conﬂict of interest statement. None declared. REFERENCES 1. Burgess,R.R., Travers,A.A., Dunn,J.J. and Bautz,E.K. (1969) Factor stimulating transcription by RNA polymerase. Nature, 221, 43–46. 2. Juang,Y.L. and Helmann,J.D. (1994) A promoter melting region in the primary sigma factor of Bacillus subtilis. Identiﬁcation of functionally important aromatic amino acids. J. Mol. Biol., 235, 1470–1488. 3. Panaghie,G., Aiyar,S.E., Bobb,K.L., Hayward,R.S. and de Haseth,P.L. (2000) Aromatic amino acids in region 2.3 of Escherichia coli sigma 70 participate collectively in the formation of an RNA polymerase-promoter open complex. J. Mol. Biol., 299, 1217–1230. 4. Rong,J.C. and Helmann,J.D. 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