918 Nucleic Acids Research, 2011, Vol. 39, No. 3 Figure 3. The low sequence selectivity of sA. (A) The nucleotide sequence of non-template strand DNA of the G3b promoter from 115 to +67. The bases of the 35 element, TG motif and 10 element are underlined. (B) The footprinting patterns of sA and sA-RNAP on the template and non-template strand DNA at the designed temperature (20, 25 or 37 C). The concentrations (mM) of sA used for the footprinting assay are shown under the horizontal lines and the concentration of sA-RNAP used is 100 nM. The numbers shown on the left of each panel are positions relative to A the transcription start site of the G3b promoter. The dotted lines indicate the –10 and –35 regions of the G3b promoter DNA protected by s . at either 10, 35 or 10/ 35 elements were compared. The footprints of sA at both 10 and 35 elements were observed on both strands of each promoter DNA in the A presence of a higher concentration (20 mM) of s , no matter whether there is a promoter mutation or not (Figure 4). However, the footprints of sA at both 10 and 35 elements (as indicated by dotted line) were only observed for the Wt and the Mu promoter DNA with base substitutions at 35 element as the sA concentration was decreased to 10 mM. No signiﬁcant footprint of sA at 10 element was detected for the G3b promoter DNA with 10 or 10/ 35 substitutions under the same condition. This result supports the idea that the in vitro sA-promoter interaction is 10 speciﬁc. Furthermore, we compared the promoter DNA-binding activities of the Wt- and the Mu-sA of which Gln-196 and Thr-199, which are assumed to be critical for the sequence-selective interaction of sA-RNAP with 12T of promoter DNA (51,53), are replaced with Ala. To achieve accurate comparison, we puriﬁed the Wt- and Mu-sA overproduced in E. coli in a soluble form and analyzed their promoter DNA-binding activities. A rela- tively weaker afﬁnity of the Mu-sA, comparing the Wt, for promoter DNA was observed in EMSA (Figure 5A). Moreover, the footprints of the Mu-sA at 10 and 35 elements on both strands of the G3b promoter DNA were undetectable, whereas those of the Wt-sA were easily detected under the same condition (Figure 5B). More im- portantly, the 10 preference or 10 speciﬁcity became insigniﬁcant for the Mu-sA as revealed from competition assay using the Wt G3b promoter DNA or the G3b promoter DNA containing base substitutions at 35 element as a competitor (Figure 5C). The requirement of the conserved amino acid residues in Region 2.4 of sA (Figure 5) and conserved bases in promoter 10 element for efﬁcient sA-promoter DNA interaction (Figure 4)