PURIFICATION OF ARCA AND ANALYSIS OF ITS SPECIFIC INTERACTION WITH THE PFL PROMOTER-REGULATORY REGION

ArcA is one of several transcription factors required for optimal anae robic induction of the pyruvate formatelyase (pfl) operon. To aid the study at the molecular level of the interaction of ArcA with the pfl p romoter-regulatory region we developed a procedure for the isolation o f ArcA. The purification of ArcA involved chromatography in heparin ag arose, hydroxylapatite and Mono-Q matrices and delivered a protein tha t was > 95% pure. Gel retardation assays demonstrated that ArcA bound specifically to the pfl regulatory region. Three distinct ArcA-DNA com plexes could be resolved depending on the ArcA concentration used. Thi s finding suggested that either multiple ArcA-binding sites are presen t in the regulatory region or that ArcA can oligomerize at one or more sites. The DNA-binding activity of ArcA could be increased an estimat ed 10-fold by prior incubation of the protein with carbamoyl phosphate , suggesting that phosphorylation activates DNA binding or oligomerisa tion. DNase I footprint analyses identified four sites that were prote cted by ArcA from cleavage. Two of these sites spanned the transcripti on start site and -10 regions of promoters 6 and 7, while a third site partially overlapped the characterized binding site of integration ho st factor (IHF). ArcA exhibited the highest affinity for a stretch of DNA located between the IHF site and the transcription start site of p romoter 7. These results are congruent with the hypothesis that a high er-order nucleoprotein complex comprising several proteins, including ArcA, is required to activate transcription from the multiple promoter s of the pfl operon.