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

• Published in: Anaerobe 2013-10, Vol.23, p.62-69
• Main Authors: Katayama, Seiichi, Ishibashi, Kotaro, Gotoh, Kazuyoshi, Nakamura, Daisuke
• Format: Article
• Language: English
• Subjects: Clostridium perfringens; Escherichia coli
• ISSN: 1075-9964
• DOI: 10.1016/j.anaerobe.2013.06.007

Three phased A5-6-tracts lie upstream of the promoter of plc encoding the I--toxin (phospholipase C) of Clostridium perfringens. The I- subunits of C. perfringens RNA polymerase bind directly to the phased A-tracts via the C-terminal domain of the I- subunit (I-CTD). To identify the amino acid residues involved in the binding of C. perfringens I- subunits to the phased A-tracts, 27 amino acid residues in C. perfringens I-CTD were substituted with alanine. The affinities of the mutated I- 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 I- subunits [Kd was 6.1 (A-0.3) A 10a8 M], and that Arg261, Asn264, Gly292 and Lys294 in C. perfringens I-CTD were critical for the binding to the phased A-tracts. The topology of these amino acid residues on the predicted structure of C. perfringens I-CTD indicated a contact path with the phased A-tracts that was similar to that of Escherichia coli I-CTD with the upstream (UP) element. On the other hand, SPR analyses at different temperatures (15, 25 and 37 degree C) indicated that the affinity of the C. perfringens I- subunits for the phased A-tracts increased in a low-temperature-dependent manner, whereas that of the E. coli I- 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 I- subunit.