FIGURE 2.
A-rich sequence required for δ function. A, nucleotide sequence of the mutant spo0B promoter DNA fragments (−52/+1). The rectangular box represents the −10 promoter element. The putative δ binding site is underlined. The mutated bases are highlighted. B, in vitro transcription assay: 400 nm RNAP holo, 100 nm spo0B mut1 promoter DNA were used in the absence and presence of δ. Run-off transcript size is 57 nt. Each experiment was repeated three times and the mean of the relative amount of transcript at each concentration of δ with respect to the amount in the absence of δ were plotted as a bar graph (shown below of each panel). C, same as in B, but for the spo0B mut2 promoter DNA. D, in vivo recombinant reporter assay: same as in Fig. 1D but with the pFPVmCherry-spo0B mut2. E and F, binding of δ to A-rich DNA fragments: fluorescence anisotropy assay, and 20 nm TMR-labeled δ, was added with different spo0B promoter derivatives. Fluorescence anisotropy of the labeled δ was monitored at excitation 540 nm and emission 580 nm. Each data set represents mean of three replicates. The Kd values of δ to A-rich DNA template was estimated using the sigmoidal function.