Nucleic Acids Research, 2011, Vol. 39, No. 3 917 Figure 2. sA binds preferentially to the promoter 10 element. (A) Mutations of the G3b promoter DNA. The sequences of the 10 element, TG motif and 35 element of the Wt G3b promoter are underlined. 10, TG, 10/TG, 35, TG/ 35 and 10/ 35 indicate the six promoter mutations used for competition assay. Base substitutions in each Mu promoter are italicized. (B and C) Fractional retention of the sA-promoter DNA complex as a function of increasing molar ratio of competitor DNA to 32P-labeled G3b promoter DNA. In the EMSA-based competition assay (B), 8 mM sA and 1 nM 32P-labeled G3b promoter DNA were used to generate the sA-G3b promoter DNA complex. The competitor DNA used to challenge the sA-G3b promoter DNA complex was shown above the horizontal lines. The molar ratios of 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 as indicated under the horizontal lines. In analysis of the correlation between the fractional retention of the preformed binary complex and the log [Competitor]/[Labeled G3b promoter DNA] (C), 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. G3b promoter DNA, to challenge the preformed sA-Wt G3b promoter DNA complex suggested that the sA also binds the G3b promoter DNA with low sequence selectivity. The low sequence selectivity of pA in vitro The low sequence selectivity of sA was conﬁrmed by DNase I footprinting which probes the protein-DNA interaction in a time- and population-averaged manner. This assay was performed under various temperatures (20, 25 and 37 C) and sA concentrations (5, 10 and 20 mM), where both partial and complete titrations of the labeled G3b promoter DNA (spanning from 115 to +67 of the promoter) occurred. The speciﬁc interaction of sA-RNAP with G3b promoter DNA was also examined for reference. The speciﬁc footprint of sA-RNAP on the template strand DNA spanned from about 70 to +1 at 20 C, and extended downstream into +17 at 25 and 37 C (left panel of Figure 3B). Downstream extension of the speciﬁc sA-RNAP footprint on the non-template strand DNA was also observed as binding temperature was increased from 20 C ( 42 to +10) to 37 C ( 42 to +20) (right panel of Figure 3B). Similar footprint extension was reported for E. coli RNAP on various promoters (49,50). Different from the above observations, the footprints of sA on both strands of the G3b promoter DNA were only detected at lower temperatures (20 and 25 C) with sA concentration higher than 10 mM (Figure 3B). The presence of complete or nearly complete protection of the 10 region as well as partial protection near the 35 region supported the idea that sA binds preferentially at least to the 10 element of G3b promoter (Figure 2C). Moreover, the DNA protected by sA was much wider than that protected by sA-RNAP in both the upstream and downstream regions of the core promoter. These results indicated that, despite the preferential binding for 10 element, the sA also binds DNA with low sequence selectivity. The low sequence selectivity of sA could be substantiated by the facts that the sA has a similar afﬁnity for the G3b promoter DNA and the tgbp3 non-promoter DNA which is relatively A-T rich (Supplementary Figure S3B) and that the full-length tgbp2 cDNA is able to compete with the Wt G3b A promoter DNA for s , albeit less efﬁciently (Figure 2B and C). Similar observations were reported for the B. subtilis sA fragment lacking Region 1 (51) and the E. coli GSTs(360) (52). The promoter-speciﬁc interaction of pA is speciﬁc in vitro 10 To conﬁrm that sA binds preferentially to the promoter 10 element or that the sA-promoter interaction is 10 speciﬁc in vitro, the footprinting patterns of sA on the Wt and the three G3b promoter DNA with base substitutions