FIG. 2.
Binding of ComA∼P and RNAP to the srf promoter as observed using SPPR analysis. (A) Biotinylated srf or mutant srf promoter DNA bound to streptavidin beads was combined with RNAP and/or ComA. Bound protein was analyzed by SDS-polyacrylamide gel electrophoresis as outlined in Materials and Methods. WT, box 2 mutant, and −35 mutant srf promoter DNA was used in the indicated reactions. Protein concentrations: RNAP, 0.2 μM; ComA, 2 μM; Spx, 10 μM. (B) Ethidium bromide-stained DNA on a 1% agarose gel. Biotinylated DNA fragments of WT, box 2 mutant, and −35 mutant DNA were applied to the streptavidin beads and extracted with phenol-chloroform from streptavidin agarose beads. (C) Effect of Spx on RNAP and ComA binding to srf promoter DNA as determined by SPPR analysis. RNAP of WT and rpoAcxs-1 (Cxs-1) strains was used in the reactions. ComA∼P phosphorylated by acetyl phosphate and Spx were added in the concentrations indicated. (D) SPPR reactions with purified αCTD and ComA, untreated or treated with Spx. Amounts of proteins in reactions are indicated. The SPPR method is described in Materials and Methods.