Mode of binding of RNA polymerase α subunit to the phased A-tracts upstream of the phospholipase C gene promoter of Clostridium perfringens

•Three phased A-tracts upstream of plc promoter of Clostridium perfringens.•The Kd value between phased A-tracts and α subunit of RNA polymerase.•The amino acids involved in the binding of α subunit to phased A-tracts.•A contact path of the αCTD of RNA polymerase with the phased A-tracts.•The affinity of the α subunit to the phased A-tracts is low-temperature-dependent.

Three phased A5-6-tracts lie upstream of the promoter of plc encoding the α-toxin (phospholipase C) of Clostridium perfringens. The α subunits of C. perfringens RNA polymerase bind directly to the phased A-tracts via the C-terminal domain of the α subunit (αCTD). To identify the amino acid residues involved in the binding of C. perfringens α subunits to the phased A-tracts, 27 amino acid residues in C. perfringens αCTD were substituted with alanine. The affinities of the mutated α subunits for the phased A-tracts were examined by gel shift assays and surface plasmon resonance (SPR). The SPR analyses revealed that the phased A-tracts themselves facilitated a complex formation between the phased A-tracts and C. perfringens α subunits [Kd was 6.1 (±0.3) × 10−8 M], and that Arg261, Asn264, Gly292 and Lys294 in C. perfringens αCTD were critical for the binding to the phased A-tracts. The topology of these amino acid residues on the predicted structure of C. perfringens αCTD indicated a contact path with the phased A-tracts that was similar to that of Escherichia coli αCTD with the upstream (UP) element. On the other hand, SPR analyses at different temperatures (15, 25 and 37 °C) indicated that the affinity of the C. perfringens α subunits for the phased A-tracts increased in a low-temperature-dependent manner, whereas that of the E. coli α subunit for the UP element did not. This suggests that the phased A-tracts may not simply be a subset of the UP element, and that they show specific binding activity with the RNA polymerase α subunit.